Glyphosate applied to glyphosate-resistant (RR) cotton varieties after the four-leaf stage can decrease boll retention resulting in severe yield reductions. Enhanced glyphosate-resistant cotton (RR Flex), released for commercial use in 2006, offers a wider window of glyphosate applications without the risk of yield loss. However, no data exist regarding the effect of glyphosate application, especially late season applications, on fruit partitioning in RR Flex cotton. The objective of this research was to determine the effect of glyphosate rate and application timing on RR Flex cotton yield and fruit partitioning compared with current RR cotton. Studies were conducted during a 3-yr period (2004 to 2006), throughout the cotton growing regions of Mississippi. Roundup Ready (ST 4892 Bollgard/Roundup Ready [BR]) and Roundup Ready Flex (Mon 171 Enhanced Roundup Ready and ST 4554 Bollgard II/Roundup Ready Flex [B2RF]) cotton was planted, and glyphosate was applied at various rates and cotton growth stages. Data were collected using box mapping, a technique designed to depict yield partitioning on a cotton plant. RR Flex cotton yields were unaffected by glyphosate application timing or rate. Yields for ST 4892 BR were affected by application timings after the sixth leaf. ST 4892 BR had increased yield partitioning to position-three bolls and upper nodes with later application timings of glyphosate. Increases in seed cotton partitioned to higher nodes and outer fruiting positions were unable to compensate for fruit shed from innermost, lower fruiting sites. These data indicate that RR Flex cotton has excellent tolerance to late-season glyphosate applications.