Bovine leukemia virus (BLV) causes enzootic bovine leucosis (EBL) and is responsible for substantial economic losses in cattle globally. However, information in Africa on the disease is limited. Here, based on clinical, hematological, pathological and molecular analyses, two clinical cases of EBL were confirmed in a dairy cattle herd in Zambia. In contrast, proviral DNA was detected by PCR in five apparently healthy cows from the same herd, suggesting subclinical BLV infection. Phylogenetic analysis of the env gene showed that the identified BLV clustered with Eurasian genotype 4 strains. This is the first report of confirmed EBL in Zambia.