Background <p>Pearl millet is a nutritionally rich, climate-resilient cereal, but its large-scale utilization is constrained by rapid rancidity in flour, caused by lipid hydrolysis and oxidation. While nuclear variation for rancidity traits is studied, the contribution of cytoplasmic genomes remains less understood. This study assessed the influence of male-sterile (A1) and maintainer (B) cytoplasms on rancidity-associated traits in hybrids derived from iso-nuclear A/B pairs crossed with diverse restorers.</p> Results <p>A total of 64 hybrids were evaluated across two locations for alcoholic acidity, and activity of enzymes - lipase, lipoxygenase and peroxidase. Analysis of variance revealed significant effects of A-lines, explaining the largest share of variability (15.9–47.7%). Cytoplasmic effects were non-significant for alcoholic acidity but highly significant for enzymatic activities, contributing up to 27.8% of total variation in peroxidase. A-cytoplasm consistently enhanced lipase, lipoxygenase and peroxidase compared to B-cytoplasm, suggesting greater predisposition to rancidity. Strong cytoplasm × nuclear interactions were evident, with A-line × cytoplasm effects accounting for up to 24% of variation. Three hybrid nuclear backgrounds (IPMA 287 × 1138R, IPMA 267 × 1142R and IPMA 267 × 1233R) in A1-cytoplasm showed favourable rancidity profiles with reduced enzyme activities, highlighting scope for further exploitation.</p> Conclusions <p>Cytoplasmic background significantly influences rancidity-related enzymes in pearl millet, with A1-cytoplasm linked to faster lipid degradation. These findings emphasize the need to consider genetic background × cytoplasm interactions in breeding strategies to reduce rancidity in pearl millet.</p>