Listeria monocytogenes is a foodborne pathogen that can cause invasive illness in humans and farm animals. It is frequently isolated from dairy products and poultry. However, there have been few literatures on the genetic diversity and virulence potential of L. monocytogenes from freshwater animal. Thirty-nine L. monocytogenes strains from crayfish were isolated and identified in this study. Molecular subtyping and polymorphism of each isolate were analyzed by multilocus sequence typing (MLST). MLST divided the isolates into eight sequence types (STs), six of which from crayfish were the same with the isolates from environment and clinic. PCR detection of the eight genes related to virulence and multiplex PCR for serotyping showed that the eight virulence factors were present in the isolates and all the isolates belonged to four major L. monocytogenes serotype groups (1/2a, 1/2b, 1/2c, and 4b) frequently isolated from patients. In vivo pathogenicity of isolates was also evaluated in murine model and survival curve of infected mice suggested that ST1, ST4, and ST9 isolates were as virulent as the reference strain EGDe. This study provides preliminary insights into the genetic diversity of L. monocytogenes from crayfish and the genetic correlation between crayfish and clinical L. monocytogenes isolates. The results indicate the contamination in aquaculture could be the source of Listeria contamination and the isolates are likely to cause human listeriosis.