Cytoplasmic male-sterility (CMS) is the primary source for the production of commercially viable onion F1 hybrids. The molecular interplay of the cytoplasmic-nuclear genes propels the restoration of fertility in the CMS system, making it cost-effective and stable. The use of the molecular markers that can determine the cytoplasm type and nuclear genotype reduces the amount of time and labour required. This study characterized the morphology of male sterile and fertile plants based on anther colour and pollen viability. Additionally, the molecular characterization of the organellar DNA differentiating cytoplasm and nuclear genotypes in 35 commercially grown open-pollinated varieties of Indian short-day onion was attempted. Our results revealed that morphological and microscopic observations for the identification of male sterile and fertile plants were not 100% corroborative. Markers located in the chloroplast (accD) and mitochondrial DNA (MKFR) revealed that Indian cultivars exhibited a greater frequency of N (normal) cytoplasm, lower frequency of S (sterile) cytoplasm and no occurrence of T (sterile) cytoplasm. All three markers viz., AcPMS1, AcSKP1, and jnurf13 revealed that 93 to 99% of the plants of all the varieties had homozygous recessive (msms) alleles at the Ms locus. The OPT marker classified the plants as having 38% msms, 39% Msms and 21% MsMs genotypes and needs further investigation. This underscores the pressing need for additional markers to precisely discern the Ms locus, facilitating the identification of male sterile and maintainer plants within open-pollinated populations of Indian short-day onions. Notably, male sterile cytoplasm was identified in eight commercial varieties, marking a pioneering revelation in Indian onion cultivation.