Background: A broad variety of infections, ranging from skin infections to infective endocarditis can be caused by Staphylococcus lugdunensis. Bacterial virulence is often related to virulence genes, so we sought to investigate the relationship between virulence genes and the pathogenicity of S. lugdunensis and to explore an appropriate typing method to distinguish different pathogenic phenotypes of S. lugdunensis. Methods: We describe the distribution of several virulence genes in different infection types in an attempt to find the relationship between virulence genes and pathogenicity. Subsequently, we make the Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) dendrogram and fbl-typing were performed using BioNumerics software, tried to compare the correlation between different methods and the different infectious diseases, and antimicrobial resistance of the strains, in order to obtain the epidemic characteristics and antimicrobial resistance information of S. lugdunensis based on a molecular approach. Results: The results of virulence genes showed that the seven virulence genes we have described existed in most strains, and there was no significant correlation between virulence gene distribution and infection type. Compared with the MALDI-TOF MS dendrogram, we found that fbl-typing could better correspond to the pathogenic phenotype, with better recognition and reproducibility. In the phylogenetic tree constructed in the fbl R-region, we found a tendency for some infection types to be distributed in clusters, new type 3 was the most dominant fbl-type, followed by fbl47b. Bone and joint infection isolates and ear infection isolates were significantly clustered together, in addition, all the oxacillin-resistant isolates were concentrated in fbl-type fbl45j and fbl47b. Conclusions: In this study, we found no significant correlation between virulence genes from S. lugdunensis isolates and the site of infection. The fbl-typing has the characteristics of convenient operation, low cost, high repeatability, and is preferable to indicate the pathogenic phenotype. Based on fbl-typing, we described the epidemiological characteristics of S. lugdunensis in a hospital and supplemented the data for fbl-typing. We recommend that fbl-typing method be extended and supplemented.