Abstract <p>Taxonomic ambiguities within the subfamily Smiliogastrinae, particularly involving the genera <i>Puntius</i> and <i>Pethia</i>, have led to ongoing debates about species classification and phylogenetic relationships. In this study, we used otolith shape and morphometric analysis to differentiate the four species of subfamily Smiliogastrinae (<i>Puntius chola</i>, <i>Puntius sophore</i>, <i>Pethia conchonius</i>, and <i>Pethia ticto</i>). We collected 134 fish samples from the local fish market of Aligarh. Otolith shape was analysed through wavelet coefficients, revealing significant differences among the species (<i>p</i> = 0.001) by PERMANOVA. The Kruskal−Wallis and the post-hoc test identified significant differences between the four species in several otolith morphometric parameters. Canonical analysis of principal coordinates indicated that the first two axes explained 61.3 and 25.3% of the variance for wavelet coefficients and 93.2 and 6.5% for otolith morphometric indices. Canonical analysis scores tested by ANOVA-like permutation test revealed significant separation (<i>p</i> = 0.001). The wavelet coefficients showed a higher classification rate (89.55%) than the otolith morphometric parameters (81.34%) through cross-validation classification. UPGMA cluster analysis revealed two major groups, indicating close relationships between <i>Pethia conchonius</i> and <i>Pethia ticto</i>, and between <i>Puntius chola</i> and <i>Puntius sophore</i>. These findings support the use of otolith morphology as a complementary tool for resolving taxonomic uncertainties in Smiliogastrinae.</p>