Marker assisted selection (MAS) using mitochondrial DNA based marker cytochrome b (cob) was integrated with phenotypic evaluation to isolate male sterile and maintainer lines from open-pollinated onion varieties adapted to North Indian agro-climatic region. Cytotype (N/S) determination by cob marker followed by morphological and microscopic study of pollen discovered male sterile plants ( Smsms ) at frequencies of 0.015 in Punjab Naroya, 0.020 in Punjab Selection, and 0.006 in Punjab White. The progeny scoring of test-crosses between male sterile and N-cytoplasmic plants isolated the maintainers (Nmsms) at frequencies of 0.133 in Punjab Naroya, 0.231 in Punjab Selection and 0.182 in Punjab White. As a novel approach, Trait Recovery Programme was demonstrated to reduce the population size... required to recover a male sterile plant by 91.08% in Punjab Naroya, 92.99% in Punjab Selection and 97.66% in Punjab White. For recovering a maintainer, 10% reduction in Punjab Naroya and 9.10% in Punjab Selection was calculated. However, no reduction was observed in Punjab White. This analysis also validated that in a randomly mating onion population, frequency of recessive ms allele squared is equal to the frequency of male sterile plants among S-cytotype and frequency of maintainers among N-cytotype ( fms2 = fSmsms/fS = fNmsms/fN).