ABSTRACT Cytoplasmic male sterility (CMS) is used for hybrid seed production in onions and S cytoplasm is widely utilized due to its better stability and simple inheritance. In CMS‐S, male fertility is restored by the single dominant allele at the nuclear male fertility restoration locus ( Ms ). Onion plants with S cytoplasm and homozygous recessive genotype at Ms locus are male sterile (S msms ) and these are sexually propagated by crossing with a male fertile plant having N cytoplasm and homozygous recessive alleles (N msms ) at fertility restoration locus. To differentiate sterile and fertile plants (cytotype and fertility restoration identification), through conventional morphological and crossing approaches is a time‐consuming and laborious process but these limitations can be overcome with marker‐assisted selection. In this study, we developed high throughput Kompetitive Allele‐Specific PCR (KASP) markers for cytotype (N/S) and fertility restoration identification. KASP assay has predicted N/S cytoplasm with 100% accuracy in different Indian populations. In addition, we utilized a unique SNP marker for Allium galanthum cytoplasm that successfully distinguished Gal (CMS) from normal (N) and sterile (S) cytoplasm within Indian populations. To identify the Ms locus, we developed KASP assays, which exhibit a high level of accuracy (~97%) in determining Ms locus. To our knowledge, this is the first report on the development and utilization of KASP markers for cytotype (N and S) and fertility restoration differentiation in Indian onion populations. The newly developed KASP markers for cytotype (N and S) and fertility restoration locus will be useful for breeders to screen a large number of plants in a cost and time‐effective manner to isolate A and B lines to accelerate heterosis breeding programme.