We investigated the incidence of human T-cell leukemia virus type I (HTLV-1) infection in a total of 17 regions in four countries, including 13 regions in Japan, as well as Korea (Seoul and Busan), China, and Vietnam, by testing breast milk using a particle agglutination assay (PA) and line immunoassay (LIA). Among 266 samples from Japan, 24 (9.0%) were positive on PA and 3 (1.1%) were positive on LIA. Among 50 samples from Seoul, 2 were positive on PA and 1 was positive on LIA. In contrast, all 50 samples from Busan were negative on both tests, suggesting the maldistribution of HTLV-1 infectants in South Korea. The numbers of positive samples were 2/91 on PA and 1/91 on LIA for China and 1/88 on both PA and LIA for Vietnam. In China, one sample with a high probability of HTLV-2 infection was identified by LIA and synthetic peptide enzyme-linked immunosorbent assay (ELISA). We examined HTLV-1 antibody in breast milk samples using commercially available test kits, suggesting the existence of HTLV-1 carriers in endemic areas in Southeast Asia and an HTLV-2 infectant in China. As a part of human ethno-epidemiological research, these results constitute valuable epidemiological data. Further studies on the sensitivity, specificity, and reliability of assays using antibodies to HTLV-1 and 2 in breast milk will be necessary for large-scale epidemiological surveys of HTLV infection.Key words breast milk; human T-cell leukemia virus type II; line immunoassay; particle agglutination assay Adult T-cell leukemia (ATL) is a malignant CD4-positive T-cell neoplasm caused by infection with human T-cell leukemia virus type I (HTLV-1). Because HTLV-1 is a retrovirus, its genomic RNA is incorporated into target cells, and DNA is incorporated into the host genome as a provirus via reverse transcriptase.