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Date: 20 September 2000
Summary
Semi-final 1
| Rank | Name | Nation | Time |
| 1 | Dara Torres | USA | 55.02 |
| 2 | Martina Moravcova | SVK | 55.06 |
| 3 | Wilma van Rijn | NED | 55.28 |
| 4 | Therese Alshammar | SWE | 55.31 |
| 5 | Susan Rolph | GBR | 55.69 |
| 6 | Sarah Ryan | AUS | 55.93 |
| 7 | Laura Nicholls | CAN | 55.94 |
| 8 | Alena Popchanka | BLR | 56.40 |
| Rank | Name | Nation | Time |
| 1 | Inge de Bruijn | NED | 53.77 |
| 2 | Jenny Thompson | USA | 54.40 |
| 3 | Helene Muller | RSA | 55.24 |
| 4 | Sumika Minamoto | JPN | 55.62 |
| 5 | Karen Pickering | GBR | 55.71 |
| 6 | Rania Elwani | EGY | 55.85 |
| 7 | Louise Joehncke | SWE | 55.94 |
| 8 | Sandra Voelker | GER | 55.97 |