Human T-lymphotropic virus type 1 (HTLV-1) infection is an important health issue that affects a variety of endemic areas. The Khorasan province, mainly its capital Mashhad in northeastern Iran, was reported to be as one of these endemic regions. Torbat-e Heydarieh, a large city Southwest border to Mashhad with a segregated population was investigated for the prevalence and associated risk factors of HTLV-1 infection in 400 randomly selected individuals. Blood samples were tested for the presence of HTLV-1 antibodies via the ELISA method and then were confirmed by an Immunoblot test. For the presence of HTLV-1 in lymphocytes of infected subjects, PCR was performed on LTR and TAX regions. DNA sequencing of LTR fragment was also carried out to determine the phylogenetic of HTLV-1, using the Maximum likelihood method. HTLV-1 sero-reactivity (sero-prevalence) among the study population was 2% (8/400), of which 1.25% had HTLV-1 provirus in lymphocytes (actual prevalence). HTLV-1 infection was significantly associated with the age, marital status, and history of blood transfusion (P < 0.05). However, there were no statistical differences between HTLV-1 infection, and gender, surgery, and hospitalization. In regression analysis, age showed the most significant correlation with the infection (P = 0.006, OR = 4.33). Based on our phylogenetic study, the HTLV-1 prevalent sequence type of Torbat-e Heydarieh belongs to the cosmopolitan subtype A. HTLV-1 prevalence in Torbat-e Heydarieh (1.25%) is low comparing to those of both Mashhad (2-3%) and Neishabour (3.5-5%) in the province of Khorasan. Thus, traveling mobility and population mixing such as marriage, bureaucratic affairs, occupation, and economic activities could be the usual routs of HTLV-1 new wave of spreading in this segregated city.