Anemia is the major public health problem in many areas of Southeast Asia. Ascertaining anemia and defining its underlying causes are essential for providing appropriate care and management of the patients as well as the establishment of a control program. There are limited studies on anemia as well as thalassemia and iron deficiency in people who living in the border regions of Thailand, Lao PDR and Cambodia. This study provided more comprehensive and meaningful data of iron deficiency thalassemia and hemoglobinopathies in the region. The prevalence of anemia, iron deficiency and thalassemia were examined in 4 districts along the border regions of Thailand, Lao PDR and Cambodia. Blood specimens were collected from subjects aged 15–18 years in 4 areas; 36 in Kantharalak, Si Sa Ket Province, 98 in Nam Yuen, 109 in Nam Khun and 128 in Na Chaluai, Ubon Ratchathani Province. RBC parameters, serum ferritin levels, Hb and DNA analyses for the identification of common thalassemia genes in Thailand were investigated. Measurement of C-reactive protein was performed to exclude false negative result of iron deficiency. The prevalence of anemia was found to be 25.1%. Iron deficiency was observed to be 14.8%. Various types of thalassemia were identified in 67.7% of the subject population. The carrier frequencies were 41.8% for Hb E, 3.8% for α0-thalassemia, 0.5% for β-thalassemia and 45.8% for α+-thalassemia. The proportions of iron deficiency, thalassemia and combined thalassemia and iron deficiency among anemic subjects were 8.6, 62.4 and 21.5%, respectively. The results indicate that thalassemia and hemoglobinopathies are a significant health burden in the border regions of Thailand, Lao PDR and Cambodia. This information will serve as a more practical basis for developing genetic counseling for the long-term effort to reduce the burden of severe thalassemia disease in the region.