Background Vibrio harveyi is an important pathogen responsible for severe economic losses in the aquaculture industry worldwide. Bacterial pathogenicity and antibiotic resistance which are of concern for environmental safety and public health exhibit significant spatial distribution patterns. In this study, we determined the prevalence, antibiotic resistance profile, and antibiotic resistance and virulence factor genes of V. harveyi isolated from diseased marine fishes in south coastal China (incl. Hainan, Guangdong and Fujian Province), which can help understand the driving environmental factors of bacterial virulence and antibiotic resistance changes. Results A total of 107 V. harveyi strains were identified by multilocus sequence analysis of 16S rRNA- toxR Vh - rctB (incl. 2 from Fujian, 52 from Hainan, and 53 from Guangdong). Nine typical virulence genes highly presented among isolates with higher average number in Hainan (7.39±0.24) than in Guangdong (6.91±0.28). Five atypical virulence genes were detected in some isolates. Specially, flaC and vvh were detected in more than 60% of the isolates. Its average number was significantly higher in Hainan (2.30±0.20) than in Guangdong (1.70±0.10). V. harveyi isolates were generally resistant (>50%) to vancomycin, amoxicillin, and midecamycin, showed moderate resistant (10%~50%) to tobramycin, gentamicin, furazolidone, rifampicin, tetracycline, and trimethoprim-sulfamethoxazole, but were nearly susceptible (<10%) to other antibiotics tested. Aac(6′)-Ib , tetW , and sul2 were detected in more than 10% isolates. The mobile genetic elements (MGEs) of int and intI1 were presented in 14.95% and 24.30% isolates, respectively. Both the average number of antibiotic resistance and antibiotic resistance genes were higher in Hainan (5.25±0.27 and 1.11±0.15) than in Guangdong (3.87±0.21 and 0.75±0.10). Conclusion This study demonstrated that multiple antibiotic resistances V. harveyi was highly prevalent with being the potential pathogen of marine fishes in South China Sea which requires particular attention. Moreover, both virulence genes and drug resistance were presence more / stronger in Hainan than in Guangdong which suggests that warming temperature and antibiotics pollutants probably enhance antibiotic resistance and bacterial infection. In the future, laboratory chamber experiment are recommend to study the response mechanisms of bacterial virulence and drug resistance to environmental factors, especially temperature and drug pollutants.