SUMMARYDrought stress significantly affects cotton's net photosynthetic rate (A) by restraining stomatal (gs) and mesophyll conductance (gm) as well as perturbing its biochemical process, resulting in yield reductions. Despite the significant progress in dissecting effects of drought on photosynthesis, the variability observed in cotton's gm, and the mechanisms contributing to that variability under dynamic drought stress conditions are poorly understood. For that reason, a controlled‐environment experiment with two cotton genotypes (Dexiamian 1, Yuzaomian 9110), three water levels (soil relative water content: control [75 ± 5]%, moderate drought [60 ± 5]%, severe drought [45 ± 5]%), and two drought durations (10 and 31 days) were conducted. The results indicated that the cotton boll biomass was significantly decreased under 10‐day severe drought and 31‐day moderate and severe drought. Decreases in gs were later accompanied by decreases in gm and further combined with reductions in electron transport rate, as drought stress progressed in duration and severity, ultimately resulting in significant reductions in A of subtending leaf. Stomatal and mesophyll conductance constraints were the primary factors limiting photosynthesis, while biochemical constraints decreased, as drought stress progressed. Considering gm, its decline was ascribed to increases in the diffusion resistance of CO2 through cytoplasm (rcyt), under short‐ or long‐term drought, as well as to increases in leaf dry mass (LMA), and decreases in the chloroplast surface area exposed to intercellular air space (Sc/S), under long‐term drought. It was concluded that A could be enhanced, under dynamic drought stress conditions, by increasing gm through increasing Sc/S and reducing LMA and rcyt.