The genetics of phenotypic plasticity in the adaptation of freshwater colonization in anadromous fish can provide important insights into the generating of teleosts diversity and invasion niche. Here, we conducted a comparative population genomic analysis of two lineages of tapertail anchovy (Coilia nasus), the anadromous and the lake-resident populations, which complete their lifecycle independently in freshwater lakes. We tracked C. nasus during their migratory period and compared genome-wide genotyping of the freshwater population with physiological traits that colonization contributes to osmotic adaptation. Through genome scans between anadromous and freshwater populations identified osmoregulation and immunoregulatory pathways evolving by strong natural selection, whereas association mapping revealed loci that contribute to freshwater adaptation. Our findings suggest that patterns of divergence in physiological capabilities are consistent with parallel adaptation to local niches. Notably, a lot of cortisol-regulated genes (e.g., ATP1α1) appeared to mutate and dynamics expression in different forms, which enriched in the ion channels, implying that the reproductive phenomenon of osmoregulation in anadromous is critical for the adaptive divergence of freshwater environments. Taken together, these results enhance our understanding of the genetic architecture variation of teleosts adaptation to novel environments, enabling cross osmotic boundaries, which leads to population divergence and freshwater colonization.