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Rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), is a popular cold-water fish widely distributed and farmed globally. In the nineteenth century, rainbow trout were introduced into India, and since then, it has gradually spread and established itself as one of the most prevalent non-native fish. In Southern India, rainbow trout were introduced from New Zealand in 1909 in the Ooty region of the Western Ghats, followed by introductions in other coldwater regions in the Peninsular uplands such as Munnar and Kodaikanal. Continuous introductions were done from various geographical locations at different periods to increase O. mykiss stocks in the Peninsular upland regions. Despite being regularly introduced in the streams of Munnar, Ooty, and Kodaikanal of Southern India, they still have not become self-sustaining, and genetic diversity has been suggested as a potential underlying factor. Therefore, this study aimed to resolve these questions and explore the structure and origin of Southern Indian stocks by conducting a population genetic study. The Y-linked marker of the trout stocks sampled from Munnar, Ooty, and Kodaikanal was compared with that of the native populations from North America. The results showed less proportion of inter-population genetic variation, suggesting that Southern Indian stocks were derived from multiple origins of population, with a great majority of parental populations belonging to the coastal rainbow trout from North America. This study revealed no considerable genetic differences among the Southern Indian stocks and reported the major ecotype as the Steelhead trout, Oncorhynchus mykiss irideus (Gibbons, 1955). [ABSTRACT FROM AUTHOR]