Molecular identification and hybrid purity testing in nineonion hybrids along with their seven parental lines wasconducted at ICAR-Directorate of Onion and GarlicResearch, Rajgurunagar, Pune (Maharashtra). Four out often microsatellite (SSRs- Simple Sequence Repeats) markerswere found to be polymorphic. All four polymorphic markersrevealed polymorphism between the male and female parentsof one or more hybrids and was utilized for their hybriditytesting. ACM016, ACM033 and ACM078 have beenidentified as the three diagnostic markers for testing thegenetic purity of all the nine onion hybrids included in thisstudy viz.; DOGR Hy-1, DOGR Hy-2, DOGR Hy-3, DOGRHy-4, DOGR Hy-5, DOGR Hy-6, DOGR Hy-7, DOGR Hy-8and DOGR Hy-50. These 10 SSR loci amplified a total 28alleles in 9 hybrids and 7 parents with 2-4 alleles per loci.The average number of alleles per loci was found to be 2.8.The highest polymorphism information content (PIC value)was observed to be 0.7137 for the marker ACM093 and thethree potential markers ACM016, ACM033 and ACM078with PIC value 0.5874, 0.5949 and 0.6628, respectively werethe best markers that could be used to differentiate theparental lines and utilized for hybridity testing. This studyshowed that SSR are reliable and effective molecular markercapable of detecting genetic purity of onion hybrids.