To explore the genetic structure and variation of the current largemouth bass (Micropterus salmoides) population, mitochondrial D-loop sequences and 14 simple sequence repeat (SSR) markers were used in this study. A total of 175 individuals from three cultured populations (“China-Taiwan” CTW, “YouLu1” YL1, “YouLu3” YL3), one breeding population (northern subspecies, US) and one “Hybrid population” (northern subspecies♂ × “YouLu 3” ♀, HYB) were analyzed for genetic variation. The results showed that all 14 microsatellite loci could be effectively amplified, and five loci (LMB24, LMB28, LMB38, LMB39, and LMB42) showed high polymorphism (PIC＞0.5). The highest polymorphism level was detected in the US population (PIC=0.514), whereas the polymorphism levels of the other four populations were lower (0.278＜PIC＜0.359). Based on D-loop sequencing, 23 mutation sites and 23 haplotypes were detected, and the haplotype diversity of the 5 populations ranged from 0.218 to 0.882. Abundant haplotypes (n=12) were detected in the US population. The dominant haplotype (H01) was detected in the other four populations, with contributions ranging from 76.7%–85.7%. Analysis of genetic variation based on SSR and D-loop sequences showed that Nei’s genetic distance and K2P genetic distance (0.3003 and 0.012, respectively) between the US and YL3 populations were the farthest (0.300 and 0.012, respectively), which where greater than those among other populations (0.016–0.297 and 0.000–0.012, respectively). Molecular analysis of variance showed that the genetic differentiation of the five largemouth bass populations was highly significant (P＜0.01). The results of the genetic structure and haplotype network analysis suggested that relative genetic independence was exhibited in the US population, whereas similar genetic resources were found in other cultured populations. The results showed that the newly introduced US population maintained a high level of genetic diversity and showed significant genetic differentiation from the domestic breeding populations. In conclusion, selective breeding of the US population and/or their crossbreeding with other populations has better prospects for genetic improvement.