<p>Self-incompatibility (SI) is a vital mechanism for promoting out-crossing in the production of hybrid seeds, understanding the reproductive behavior of Protogyny (PG) genotypes can assist in selecting appropriate parental plants for hybrid-breeding programs, enabling the creation of novel mustard hybrid in a more systematic and logical manner. The present study was focused on genetics and identification of putative candidate genes for PG-SI in Indian Mustard. F₁s and F₂ populations were developed by crossing Pusa Tarak with PG-SI and ten normal genotypes. F₂ segregation pattern have shown that the PG-SI trait was controlled by digenic-duplicate gene action with 15:1 ratio. To understand the genetic basis of PG-SI trait in Indian mustard, we performed genotype-by-sequencing (GBS)&#xa0;of PG-SI and non-PG-SI lines developed in three different genetic background (Pusa Tarak, Pusa Bold and Pusa Mahak) using Illumina platform. Single nucleotide polymorphisms (SNPs) differentiating 3 PG-SI and 3 non-PG-SI genotypes were identified. A total of 4,88,663 SNPs and 4,01,301 SNPs found among 3 PG-SI genotypes and 3 Non-PG-SI genotypes, respectively. In this study, we identified corresponding alleles of 4 SNPs consistently associated with all protogynous (PG-SI) and non-protogynous (non-PG-SI) genotypes of <i>Brassica juncea</i>. SNPs, A10:9559612, A09:694111, B05:2480898 and A03:11891971 were co-segregated in these genotypes. We identified the putative candidate genes, namely, calcium-binding protein PBP1, serine/threonine-protein kinase and calcium-dependent protein kinase 5 associated with these identified SNPs. The present findings would enhance our understanding on the genetic basis of protogyny and self-incompatibility in <i>Brassica juncea</i>. In addition, associated SNPs and candidate genes predicted in the current study could be further useful in marker assisted breeding to transfer this trait to other genetic background and subsequently, can be useful in hybrid breeding program.</p>