Glaesserella parasuis is a bacterial pathogen that causes severe economic losses in the swine industry worldwide. In this study, 262 G. parasuis isolates from pigs with Glässer's disease obtained between 2015 and 2022 in Taiwan were serotyped, pathotyped, and analyzed for virulence genes. The most prevalent serovars were 5 (29%), 4 (27.9%), 12 (12.2%), and non-typable strains (13%). The frequency of serositis with pulmonary lesions (55%) was significantly higher than that of pulmonary lesions alone (23.3%) and serositis lesions alone (21.7%) (p <
0.001). Serovars 4, 5, 12, 13 and non-typable strains had a significantly higher prevalence in serositis with pulmonary lesions than in pulmonary lesions alone, whereas serovar 1 had higher prevalence in pulmonary lesions. The virulence gene V4, related to virulence-associated protein D, had the highest detection rate (94.8%) in pathogenic isolates, which was significantly higher than other virulence genes in serovars 4, 5, 7, 12, 13, and non-typable strains except for serovar 1. Virulence-associated protein D may be a suitable subunit vaccine antigen candidate, and more research is required to evaluate the potential for broader application. Given the limited vaccine availability restricted to serovar 5 in Taiwan, these findings provide a basis for multivalent and subunit vaccine development providing better cross-protection.