Fowl adenovirus (FAdV) has caused significant losses in chicken flocks throughout China in recent years. However, the current understanding of the genetic and pathogenic characteristics of the FAdV epidemic in southwestern China remains poorly understood. In this study, a total of 22 strains were isolated from liver samples of diseased chickens from farms in southwestern China. Phylogenetic analysis based on the hexon loop-1 gene showed that the 22 isolates were clustered into four distinct serotypes: FAdV serotype 4 (FAdV-4) (86.4%, 19/22), FAdV-2 (4.5%, 1/22), FAdV-8a (4.5%, 1/22), and FAdV-8b (4.5%, 1/22). FAdV-4 was the predominant serotype in southwestern China. Pathogenicity testing showed that the FAdV-4 serotype strain CH/GZXF/1602 and FAdV-8a strain CH/CQBS/1504 were pathogenic to chickens, with mortality rates reaching as high as 80% and 20%, respectively. The primary clinical feature observed following infection with strain CH/GZXF/1602 (FAdV-4) was hepatitis-hydropericardium syndrome, and that of strain CH/CQBS/1504 (FAdV-8a) was inclusion body hepatitis. Conversely, the FAdV-2 serotype strain CH/GZXF/1511 and FAdV-8b serotype strain CH/CQBS/1512 was not observed to be pathogenic in chickens. Then, CH/GZXF/1602 (FAdV-4) was selected for the preparation of an inactivated oil-emulsion vaccine. Immune studies on Partridge Shank broilers showed that a single dose immunization at 17 days of age could not only protect against homologous challenge with virulent FAdV-4 but also provided protection against clinical disease following challenge with the heterologous FAdV-8b virulent strain until 70 days of age. The characterization of newly prevalent FAdV strains provides a valuable reference for the development of an efficacious control strategy.