Assessing haplotypic and genotypic diversity is crucial for comprehending biological invasion dynamics and devising effective management strategies. In India, Gambusia spp. were introduced in the early twentieth century for mosquito biocontrol, but recent studies have demonstrated their detrimental impact on native biota. The present research employs molecular tools to investigate the haplotypic and genotypic diversity of Gambusia species in India. The findings reveal a greater distribution of Gambusia holbrooki and a lesser occurrence of Gambusia affinis in India. The study underscores that G. holbrooki was initially brought into India from Europe and later multiple times from other regions across the globe. Our global haplotypic network shows the presence of a single G. affinis haplotype, which is narrowly distributed in the northeastern part of India. Additionally, our population genetic analyses exhibit a low genetic variability and significant population genetic structuring among G. holbrooki populations. This study emphasizes the significance of evaluating haplotypic and genotypic diversity to develop a comprehensive understanding of the invasion history and dynamics of introduced species, thereby aiding in the development of effective management strategies that mitigate their negative impact on native biota.