| Rang | Serie | Baan | Naam | Land | Tijd | Bijz. |
| 1 | 6 | 4 | Dressel , Caeleb Caeleb Dressel | Verenigde Staten | 50.08 | Q |
| 2 | 6 | 3 | Codia , Piero Piero Codia | Italië | 51.09 | Q |
| 3 | 7 | 6 | Guy , James James Guy | Verenigd Koninkrijk | 51.16 | Q |
| 4 | 8 | 4 | Schooling , Joseph Joseph Schooling | Singapore | 51.21 | Q |
| 5 | 8 | 6 | Milák , Kristóf Kristóf Milák | Hongarije | 51.23 | Q |
| 6 | 8 | 5 | le Clos , Chad Chad le Clos | Zuid-Afrika | 51.28 | Q |
| 7 | 7 | 3 | Metella , Mehdy Mehdy Metella | Frankrijk | 51.46 | Q |
| 8 | 6 | 6 | Martins , Henrique Henrique Martins | Brazilië | 51.48 | Q |
| 9 | 7 | 5 | Czerniak , Konrad Konrad Czerniak | Polen | 51.50 | Q |
| 10 | 7 | 4 | László Cseh | Hongarije | 51.55 | Q |
| 11 | 6 | 5 | Li Zhuhao | China | 51.62 | Q |
| 12 | 7 | 2 | Irvine , Grant Grant Irvine | Australië | 51.67 | Q |
| 13 | 6 | 7 | Świtkowski , Jan Jan Świtkowski | Polen | 51.82 | Q |
| 14 | 8 | 8 | Popkov , Aleksandr Aleksandr Popkov | Rusland | 51.84 | Q |
| 15 | 8 | 2 | Morgan , David David Morgan | Australië | 51.90 | Q |
| 16 | 8 | 3 | Phillips , Tim Tim Phillips | Verenigde Staten | 51.96 | Q |
| 17 | 7 | 0 | Goosen , Mathys Mathys Goosen | Nederland | 52.12 | |
| 18 | 8 | 1 | Quah Zheng Wen | Singapore | 52.13 | |
| 19 | 5 | 6 | Sabo , Sebastian Sebastian Sabo | Servië | 52.16 | |
| 20 | 7 | 7 | Kobori , Yuki Yuki Kobori | Japan | 52.17 | |
| 21 | 7 | 8 | Martínez , Luis Luis Martínez | Guatemala | 52.18 | |
| 22 | 6 | 2 | Kusch , Marius Marius Kusch | Duitsland | 52.22 | |
| 23 | 7 | 1 | Pachomov , Daniil Daniil Pachomov | Rusland | 52.47 | |
| 24 | 4 | 1 | Ayar , Kaan Kaan Ayar | Turkije | 52.52 | |
| 25 | 6 | 1 | Grassi , Santiago Santiago Grassi | Argentinië | 52.59 | |
| 26 | 5 | 2 | Šefl , Jan Jan Šefl | Tsjechië | 52.65 | |
| 27 | 5 | 0 | Croenen , Louis Louis Croenen | België | 52.74 | |
| 28 | 7 | 9 | Carter , Dylan Dylan Carter | Trinidad en Tobago | 52.75 | |
| 29 | 8 | 7 | Sankovitsj , Pavel Pavel Sankovitsj | Wit-Rusland | 52.87 | |
| 30 | 6 | 9 | Vazaios , Andreas Andreas Vazaios | Griekenland | 52.88 | |
| 31 | 4 | 2 | Kozljoek , Artjom Artjom Kozljoek | Oezbekistan | 52.95 | |
| 32 | 6 | 8 | Lemesjko , Ljoebomyr Ljoebomyr Lemesjko | Oekraïne | 52.97 | |
| 33 | 6 | 0 | Bromer , Viktor Viktor Bromer | Denemarken | 53.01 | |
| 34 | 5 | 3 | Binnema , Josiah Josiah Binnema | Canada | 53.10 | |
| 35 | 8 | 0 | Carini , Giacomo Giacomo Carini | Italië | 53.13 | |
| 36 | 8 | 9 | Schlesinger , Marcus Marcus Schlesinger | Israël | 53.20 | |
| 37 | 4 | 4 | Moessin , Adilbek Adilbek Moessin | Kazachstan | 53.37 | |
| 38 | 4 | 3 | Liess , Nils Nils Liess | Zwitserland | 53.50 | |
| 39 | 4 | 7 | Hockin , Ben Ben Hockin | Paraguay | 53.51 | |
| 40 | 4 | 0 | Pöytäkivi , Riku Riku Pöytäkivi | Finland | 53.62 | |
| 41 | 5 | 1 | Ivanov , Antani Antani Ivanov | Bulgarije | 53.66 | |
| 41 | 5 | 4 | Sidiq , Triady Fauzi Triady Fauzi Sidiq | Indonesië | 53.66 | |
| 43 | 5 | 5 | Zirk , Kregor Kregor Zirk | Estland | 53.70 | |
| 44 | 5 | 9 | Hyland , Brendan Brendan Hyland | Ierland | 53.80 | |
| 45 | 4 | 8 | Lavado , Marcos Marcos Lavado | Venezuela | 53.97 | |
| 45 | 5 | 7 | Duškinas , Tadas Tadas Duškinas | Litouwen | 53.97 | |
| 47 | 3 | 2 | Chou Wei-liang | Chinees Taipei | 54.31 | |
| 48 | 3 | 3 | Prakash , Sajan Sajan Prakash | India | 54.46 | |
| 49 | 3 | 4 | Arias , David David Arias | Colombia | 54.48 | |
| 50 | 4 | 9 | Martínez , José José Martínez | Mexico | 54.50 | |
| 51 | 5 | 8 | Eissa , Omar Omar Eissa | Egypte | 54.59 | |
| 52 | 4 | 5 | Hoàng Quý Phước | Vietnam | 54.76 | |
| 53 | 3 | 5 | Goveia , Ralph Ralph Goveia | Zambia | 54.86 | |
| 54 | 3 | 7 | Sperandio Sánchez , Christian Christian Sperandio Sánchez | Dominicaanse Republiek | 54.98 | |
| 55 | 3 | 6 | de Silva , Cherantha Cherantha de Silva | Sri Lanka | 55.09 | |
| 56 | 3 | 0 | Ansari , Mehdi Mehdi Ansari | Iran | 55.31 | |
| 57 | 2 | 4 | Umnov , Mikhail Mikhail Umnov | Malta | 55.58 | |
| 58 | 3 | 9 | Anis , Rami Rami Anis | Onafhankelijke atleten | 55.66 | |
| 59 | 3 | 8 | Alvarez , Bryan Bryan Alvarez | Costa Rica | 55.85 | |
| 60 | 3 | 1 | Kelzi , Ayman Ayman Kelzi | Syrië | 56.59 | |
| 61 | 2 | 3 | Soammanus , Jeerakit Jeerakit Soammanus | Thailand | 56.88 | |
| 62 | 2 | 5 | Toure , Oumar Oumar Toure | Mali | 57.52 | |
| 63 | 2 | 1 | Nmor , Ifeakachuku Ifeakachuku Nmor | Nigeria | 57.90 | |
| 64 | 1 | 3 | Al-Doori , Abdullah Abdullah Al-Doori | Irak | 58.01 | |
| 65 | 2 | 2 | Jabbour , George George Jabbour | Honduras | 58.29 | |
| 66 | 1 | 5 | Al-Khulaifi , Yacob Yacob Al-Khulaifi | Qatar | 58.42 | |
| 67 | 2 | 6 | Gaskin , Hannibal Hannibal Gaskin | Guyana | 58.69 | |
| 68 | 1 | 4 | Francisco , Daniel Daniel Francisco | Angola | 58.79 | |
| 69 | 1 | 6 | Poppe , Tanner Tanner Poppe | Guam | 1:03.28 | |
| 70 | 2 | 9 | Dlamini , Simanga Simanga Dlamini | Swaziland | 1:04.02 | |
| 71 | 2 | 7 | Roberto , David David Roberto | Noordelijke Marianen | 1:06.80 | |
| 72 | 2 | 0 | Gooding , Dillon Dillon Gooding | Saint Vincent en de Grenadines | 1:07.11 | |
| 4 | 6 | Perry , Sam Sam Perry | Nieuw-Zeeland | DNS |