Premature leaf senescence negatively influences the physiology and yield of cotton plants. The conserved IDLNL sequence in the C-terminal region of AGL42 MADS-box determines its repressor potential for the down regulation of senescence-related genes. To determine the delay in premature leaf senescence, Arabidopsis AGL42 gene was overexpressed in cotton plants. The absolute quantification of transgenic cotton plants revealed higher mRNA expression of AGL42 compared to that of the non-transgenic control. The spatial expression of GUS fused with AGL42 and the mRNA level was highest in the petals, abscission zone (flower and bud), 8 days post anthesis (DPA) fiber, fresh mature leaves, and senescenced leaves. The mRNA levels of different NAC senescence-promoting genes were significantly downregulated in AGL42 transgenic cotton lines than those in the non-transgenic control. The photosynthetic rate and chlorophyll content were higher in AGL42 transgenic cotton lines than those in the non-transgenic control. Fluorescence in situ hybridization of the AG3 transgenic cotton line revealed a fluorescent signal on chromosome 1 in the hemizygous form. Moreover, the average number of bolls in the transgenic cotton lines was significantly higher than that in the non-transgenic control because of the higher retention of floral buds and squares, which has the potential to improve cotton fiber yield.