Background <p>The prevalence of Human T-Cell Lymphotropic Virus Type 1 (HTLV-1) represents a major public health challenge, threatening the safety of blood transfusions for patients globally, particularly in Yemen due to the lack of routine pre-transfusion screening for this type of virus. This study aimed to determine the prevalence and associated factors of HTLV-1 among multi-transfused patients (MTPs) in Sana’a city.</p> Methods <p>An analytical cross-sectional study was conducted among MTPs at medical treatment centers in Sana’a city. Data were collected through face-to-face interviews using a questionnaire that included sociodemographic information and predisposing factors of infection. A quantitative ELISA kit was used to detect HTLV-1 antigen in serum/plasma samples and a positive ELISA test was confirmed using real-time PCR. All patients were also screened for HIV, HBsAg and HCV. Bivariate and multivariate analysis were performed to identify associated factors.</p> Results <p>A total of 361 MTPs were enrolled. Of them, male 51.8% and aged less than 20 years 73.7%. Seroprevalence was higher in males, married individuals, and the 20–29 age group. Out of 361 samples, 12 (3.3) were initially reactive to HTLV1/2 antigen. The PCR results confirmed 10 samples (2.8%) were positive for HTLV-1. Only one patient had co-infection of HTLV-1 with HBsAg. Patients who have a low income (AOR:12.23;95%CI: 1.37-109.37), a medical history of blood transfusion of more than 30 units (AOR: 8.14; 95%CI:1.48–44.95), acupuncture (AOR:6.66;95%CI: 1.25–35.56), and dental clinic visits (OR = 5.84;95%CI:1.01–33.74) were significantly associated with HTLV-1.</p> Conclusions <p>Although the prevalence rate of HTLV-1 was not high in Sana’a city, it remains an important problem among MTPs. Therefore, HTLV-1 screening should be mandatory in blood centers, and community awareness of risk factors must be increased.</p>