Indian oil sardines, commercially and ecologically important pelagic fishes in Indian waters have not been the focus of major genetic studies as compared to their counter parts in Atlantic and Pacific oceans in spite of several reports suggesting stock complexity and intraspecific diversity. Hence, we investigated the genetic stock structure of Indian oil sardine, Sardinella longiceps using microsatellite markers by collecting a total of 768 individuals from eight locations along the Indian coast and one from Gulf of Oman over a 2 year period (2013–2015). Six polymorphic microsatellite markers revealed significant genetic differentiation between populations with the highest FST value (0.055) between Oman and Indian coastline. Within the Indian coastline another major subdivision between Mumbai & Mangalore vs. other regions was detected (FST value 0.047) which was also confirmed in Barrier analysis with the presence of two strong barriers between these eco-regions. There exist pronounced differences in oceanographic and environmental features between Gulf of Oman, Western Indian Ocean and Eastern Indian Ocean (Bay of Bengal) which may act as barriers for effective dispersal and gene flow resulting in genetic differentiation. Even though, the samples collected from Calicut, Kollam, Trivandrum, Chennai and Vizag showed the presence of admixed genotypes, the possible presence of distinct populations in some regions was evident in Bayesian analysis which needs to be confirmed further using more widespread sampling design and powerful markers. The present study provided insights into the biocomplexity and intra-specific diversity of Indian oil sardine populations, which needs to be preserved for maintaining resilience of these important fishes to climate change and habitat alterations in the Indian Ocean.