Pasteurella multocida is a Gram-negative coccobacillus from the Pasteurellaceae family, commonly residing as a commensal organism in the respiratory tracts of healthy animals. However, it possesses multiple virulence factors and can cause severe respiratory diseases. This study aimed to characterize P. multocida and its virulence genes and evaluate the immunogenicity of an inactivated vaccine of serogroup B using different administration routes. A total of 250 samples were collected from animals showing respiratory symptoms. Using 5% blood agar, 27 P. multocida isolates were obtained, and 21 (8.4%) were confirmed via PCR targeting the kmt1 gene. Nineteen virulence-associated genes were screened, categorized into outer membrane, fimbrial, somatic antigen, and iron-binding genes. The plpB, tadD, gatG, and hgbA genes were detected in both serogroup B and E isolates, whereas ompA, toxA, pcgD, latB, nctB, ppgB, natG, hgbB, and exbB were absent in all isolates. The immunogenicity of an inactivated P. multocida vaccine was evaluated in mice using subcutaneous and intramuscular routes. Subcutaneous vaccination produced a significantly higher antibody titer at 3 and 5 weeks post-vaccination with a 0.5 mL dose; in contrast, intramuscular immunization resulted in a rapid increase after booster doses. Analysis of variance (ANOVA) and Duncan’s multiple range test (p < 0.05) revealed statistically significant differences between treatments. The comparison between subcutaneous and intramuscular routes also showed a significant difference (p < 0.001). This study concludes that although P. multocida serogroup B harbors fewer virulence factors, it effectively induces an immune response in mice but fails to provide cross-protection against the local serogroup E strain.