Improvement in lint yield is vital to commercial production of glandless cotton. The objective of this study was to determine if glanded ‘Acala 1517-08’ could be converted to glandless cotton without a yield penalty by crossing breeding with an obsolete glandless cotton. From 500 F2 plants, 18 glandless individuals were selected, and their F4 lines were compared with both parents. This led to a selection of 77 F6 lines for further replicated field testing. Only five F6 lines produced 90 to 96% of Acala 1517-08 lint yield (LY), while three lines had significantly lower LY than the lower parent ‘Acala GLS’. Transgressive segregation in the negative direction was also observed for boll weight (BW) and lint percent (LP) in that four and 22 F6 lines had significantly lower values than the lower parent, respectively. Four F6 lines had significantly lower fiber length (UHM) than the lower parent Acala 1517-08, while five lines had longer UHM than the higher parent Acala GLS. Ten F6 lines had weaker fibers than the lower parent Acala 1517-08, but none had stronger fibers than Acala GLS. Most F6 lines had micronaire (MIC) between the two parents except for one with higher MIC than the higher parent and five with lower MIC than the lower parent Acala GLS. In conclusion, positive transgressive segregation for LY, LP and BW should not be expected in this Acala × Acala cross, while negative transgressive segregations occurred frequently for the three traits and fiber strength. For UHM and MIC, negative transgressive segregations occurred more frequently than positive ones. Overall, there was no transgressive segregation for fiber uniformity, elongation and short fiber content.