Growers of glyphosate-resistant (GR) cotton (Gossypium hirsutum L.) often apply glyphosate postemergence over-the-top (POST) late in the season to control escaped weeds and to increase harvesting efficiency. Labels for glyphosate products currently permit such applications only after the 20% cracked-boll stage, which is later than most growers want to apply the glyphosate. An experiment was conducted eight times in Georgia, Louisiana, and North Carolina during 2000 and 2001 to determine the effect of glyphosate applied pre-harvest prior to the 20% cracked-boll stage on fruit set and retention and yield of GR cotton. Glyphosate isopropylamine salt at 840 g acid equivalent (a.e.) ha-1 was applied POST 7 d prior to an arbitrarily determined last effective bloom date (LEBD) or 0, 7, 14, or 21 d after the LEBD. The final application generally corresponded to the 20% cracked-boll stage. Cotton was box-mapped for fruit distribution and yield prior to mechanical harvest. No differences among treatments were detected by box mapping. Treatments containing glyphosate produced yields similar to the non-treated control, but application 7 d before or 0 or 7 d after the LEBD reduced yield compared with application 21 d after the LEBD. These results indicate that pre-harvest glyphosate application should be delayed until 14 d after the LEBD.