AbstractOtoliths are an excellent tool in studies on ecological connectivity of fish species populations. Opsanus beta is an invasive species introduced on the Brazilian coast, but not native from the Gulf of Mexico. The present study aimed to compare the otolith contours of specimens collected in Mexico (Celestún, CEL) and in two Brazilian estuaries (Santos Bay, STB, and Paranaguá Estuarine Complex, PEC). In the laboratory, 99 otoliths were extracted, photographed and compared using wavelet analysis. The otolith contours varied between sites (39 from CEL, 26 from STB and 34 from PEC). The linear discriminant analysis correctly reclassified 87.9% of otoliths by sites, with the best reclassifications in the CEL (97.36%), followed by PEC (88.23%) and SBT (73.07%). MANOVA showed significant differences in otolith contours between sites (F = 5.37; P < 0.005). The otolith contour from CEL was significantly different from those from the PEC and SBT. However, the otolith contour of the two Brazilian estuaries did not significantly differ among them (MANOVA, P > 0.005). Our results indicate O. beta populations on the Brazilian coast are connected, and probably isolated from the Mexican population.