Bovine leukemia is a lymphoproliferative disease in cattle, which is divided into two types on the basis of their epidemiology. Enzootic bovine leukosis (EBL) is caused by the infection with a retrovirus, bovine leukemia virus (BLV), and Sporadic bovine leukosis (SBL) is not transmissible. Although many reports have provided phenotypic characterization on T-cell or B-cell lymphoma in human, that in cattle is still unknown. In this study, we investigated the phenotypes of tumor cells from cattle with lymphoma by using flow cytometry. We analyzed a total of 36 clinical cases; 4 were T-cell lymphoma, 25 were B-cell lymphoma, and 7 were other types of tumor or precancer states. By the evaluation of provirus loads and B-cell clonality, 22 out of 25 B-cell lymphoma were diagnosed as EBL. Most of the EBL cases showed major B-cell phenotypes such as CD5+, IgM+, CD21+ and CD79a+, but interestingly more than half of them were negative for CD19, which is a marker of B lymphocytes expressed throughout B-cell development. In addition, we found two types of atypical EBL cases compared with previously reported ones; one was BLV-mediated B-cell lymphoma in juvenile cattle, and the other was EBL without persistent lymphocytosis. Those features might be related to genetic background of cattle developing tumors because 16 out of 22 EBL cases were Japanese native cattle. This study has reported the novel phenotypic characters of EBL, and further analysis would be required in order to reveal a mechanism of tumor development and to identify a common marker for EBL diagnosis.