Existence of CGMS system in hot pepper is due to the rearrangements in the mitochondrial genome and is largely used in economized and pure F 1 hybrid seed production around the world. The orf456, a new ORF present at flanking region of the coxII gene at the 3’ end, was distinguished male sterile cytoplasm in hot peppers along with atp6-2gene. In the current study, eighteen pepper genotypes (nine each of A and corresponding B lines) of varied origin were used to validate with two male sterile cytoplasm (S-cytoplasm) specific sequence characterised amplified region (SCAR) markers viz., atp6-2 (875 bp) and orf456 (456 bp) and one restoration-of-fertility (Rf) locus specific marker, CRF (550 bp). The results clearly showed that the presence of CMS-S-cytoplasm and absence of restoration-of-fertility (Rf) gene in the pepper genotypes studied and is comparable with the phenotypic data. In view of the outcomes it has been reasoned that the accessible S and Rf markers available in the public domain are reproducible and can be promptly utilized for marker assisted selection (MAS) in hot pepper crop improvement program.