Background and Aims:African swine fever (ASF) is a highly contagious viral disease that causes major economic losses due to morbidity and fatality rates of up to 100% in wild boar and domestic pigs. The disease emerged in Africa in 1921 and then entered several European countries by 1957. In Indonesia, the first outbreak of ASF in 2019 in North Sumatra killed thousands of pigs and quickly spread to 10 out of 34 pig-producing provinces, including Bali and Eastern Nusa Tenggara. As no commercial ASF vaccine is available, the disease has become endemic and continues killing pigs. This study aimed to investigate the epidemiological and virological studies of ASF virus (ASFV) conducted in 2020 and 2021 by the Disease Investigation Center Regional VI of Denpasar Bali, which covers three provinces in Indonesia, including Bali, Western Nusa Tenggara, and Eastern Nusa Tenggara.Materials and Methods:A total of 5402 blood samples were sent to the laboratory to detect ASFV infection using quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay tests. The virological studies were performed by culturing local ASFV isolates obtained from field cases in primary macrophages and confirmation of viral growth by qPCR.Results:The qPCR results show that only 156/4528 (3.4%) of samples originating from Bali and Eastern Nusa Tenggara were ASFV-positive with cycle threshold value of 18 to 23, while the virus was not detected in Western Nusa Tenggara. Of 874 serum samples tested, 114 (13%) were antibody positive and were all collected from the two ASFV-affected provinces in 2020. A Bali ASFV isolate (BL21) was isolated and characterized molecularly.Conclusion:These findings suggest that during the time of sampling, ASFV was detected only in Bali and East Nusa Tenggara but not in Western Nusa Tenggara. These findings support the symptomology of ASFV reported in the two regions. Moreover, BL21 may be useful for developing subculture-attenuated vaccines using commercial cell lines. However, the current study has some limitations namely the investigation was not performed during the initial outbreak and no pathological examination of internal organs was conducted.