Roundup Ready Flex cotton was introduced commercially in 2006. Although research is available documenting Roundup Ready cotton tolerance to glyphosate, little to no data exists regarding the effect of glyphosate application on fruit partitioning in Roundup Ready Flex cotton. The objective of this research was to evaluate the effect of glyphosate application on fruit partitioning in Roundup Ready Flex cotton varieties. Five experiments were conducted at three locations throughout Mississippi in 2004 and 2005. Varieties chosen for the study were DP 117 B2RF, DP 143 B2RF, ST 4554 B2RF, and ST 6611 B2RF. Variety selection was based upon differing relative maturity among the varieties. Glyphosate was applied at a labeled rate (0.84 kg ae/ha), to all varieties, four separate times throughout the growing season. Data were collected using box mapping, a technique designed to depict yield partitioning on a cotton plant. These data indicate that yield partitioning was different among varieties but was unaffected by glyphosate application for any variety. Lower percent seed cotton partitioned to lower nodes was observed for the DP 143 B2RF and ST 6611 B2RF varieties compared to DP 117 B2RF and ST 4554 B2RF. However, DP 143 B2RF and ST 6611 B2RF partitioned more seed cotton to higher nodes, starting with node 12, compared to DP 117 B2RF and ST 4554 B2RF. These data indicate that the four varieties evaluated exhibited excellent tolerance to multiple glyphosate applications with no significant differences with respect to yield. Additionally, glyphosate application had no impact on seedcotton partitioning in Roundup Ready Flex cotton.