In recent years, the problem of biological fouling by Limnoperna fortune (Dunker, 1857) has occurred in water resources and hydraulic engineering, affecting the safety of water transmission and production of water conservancy and hydropower projects to varying degrees. The problem has attracted extensive attention from the domestic and foreign communities. This study showed that L. fortunei in the Yangtze River Basin was mainly distributed in the low altitude area of Sichuan and below. No record was found in the high-altitude area above Sichuan. Phylogenetic tree analysis utilizing COI and ITS1 genes revealed two groups of L. fortunei in the Yangtze River Basin. The A+T content of COI sequences was notably higher at 64.7% compared to the C+G content at 35.3%, with intraspecific genetic distances ranging from 0 to 0.123. The total haplotype diversity index for COI sequences was 0.893. The nucleotide diversity index was 0.069. For the ITS1 sequences, the A+T content was 45.3% and the C+G content was 54.7%, with intraspecific genetic distances ranging from 0 to 0.061. The total haplotype diversity index for ITS1 sequences was 0.679. The nucleotide diversity index was 0.028. Genetic differentiation analysis based on the COI gene yielded a coefficient of genetic differentiation of 0.844 and a coefficient of differential gene differentiation of 0.118 between the two related groups. Similarly, analysis based on the ITS1 gene resulted in a coefficient of genetic differentiation of 0.927 and a coefficient of differential gene differentiation of 0.492. The morphological characteristics and habitat analysis showed that there were significant differences in the morphological characteristics and habitats of the two ecotypes of L. fortunei in the Yangtze River basin, which can be clearly divided into two groups, i.e., Group 1 and Group 2 as shown in COI and ITS1 gene analysis. The length-height ratio, length-width ratio and ligament length of the two groups were significantly different. Group 1 mainly lived in the areas with slow flow and pebble-based bottom, showing a slow flow type. Group 2 mainly lived in the areas with fast flow and sand-based bottom, showing a rapid flow type, and had stronger adaptability to the environment. It had a wider distribution range than Group 1. This study improved our understanding of the basic biological information of L. fortunei in the Yangtze River basin, and provided a reference for the comprehensive prevention and control of L. fortunei in the basin.