Trypanosoma evansi, the causative agent of "surra" is enzootic in Iran. The current study aimed to detect T. evansi in horses from different regions of Iran using morphological, serological, and molecular methods. In 2021, 400 blood samples were collected from horses in eight regions. Eighty horses showed clinical signs such as cachexia (n = 64), fever (n = 36), foot edema (n = 40), and abdominal edema (n = 32), and 320 horses appeared healthy. All samples from the studied regions were evaluated for the presence of trypanosomes using direct analysis of blood smears, mercuric chloride, and PCR-based tests. In total, 12% (95% CI: ± 3.1%), 21% (95% CI: ± 3.9%), and 21% (84) of animals were positive for Trypanosoma in microscopic, serologic, and molecular analyses, respectively. All animals positive for SSU rDNA PCR were from Qom, Semnan, and Golestan regions. Further molecular analyses on 84 PCR-positive horses revealed that 29 horses scored positive in PCR using primers of trypanozoon species and 5 scored positive in PCR using primers of Trypanosoma evansi type A. All samples (n = 5) were from Qom region. The 205-bp fragments of T. evansi RoTat 1.2VSG (accession numbers: ON017789-93) analyzed and compared to other isolates sequence from GenBank BLAST search. It has close similarities with isolates from Pakistan, Egypt, Malaysia, Kenya, and India. Data herein demonstrated that horses from Iran were at high risk of T. evansi infection. Comprehensive control programs, such as those based on the application of repellants and traps, and also, compliance with quarantine standards are recommended for minimizing the risk of the infection.