Salmonella pullorum (S. pullorum) seroprevalence was determined, followed by isolation and molecular characterization in commercial poultry in district Faisalabad, Pakistan. The bacterin vaccine was prepared from the local isolate of S. pullorum and its efficacy was evaluated in broiler chicks. For seroprevalence, a total of 384 boiler farms were screened. Seroprevalence of S. pullorum in broiler was 11.72%. The collected tissue samples (liver, spleen, and intestine) from seropositive birds were processed on different media (Xylose lysine deoxycholate agar, Salmonella shigella agar and MacConkey agar) for bacterial isolation of S. pullorum. Molecular detection was done with the help of polymerase chain reaction followed by bacterin preparation. An experimental trial was done to compare the efficacy of bacterin with the commercial vaccine (Poulvac ®, USA). A total of 120 broiler birds were divided into four groups (G1-G4) designed as G1 (negative control), G2 (infected with S. pullorum, G3 (bacterin + S. pullorum infection), and G4 (commercial vaccine (Poulvac®) + S. pullorum infection). Higher morbidity (80%) and mortality rates (60) were in group G2 as compared to the group (G3 and G4). Gross and histopathological lesions were highest in group G2 as compared to other groups. In conclusion, the locally prepared bacterin provides better protection in terms of mortality and morbidity and enhances humoral immune response in broiler birds.