Foot-and-mouth disease is a transboundary, highly contagious viral disease. The A, Asia 1, and O are the main serotypes reported in Iraq over the last 60 years; however, a recent outbreak revealed a different phenomenon. So, this study aims to detect the serotype(s) of FMDV that caused the most recent disease outbreak in buffaloes in Wassit and Dhi-Qar, Iraq. This will be done by using both clinical and molecular methods. In total, 70 buffaloes of different genders and ages were selected for the current study, performed during August and September 2023. All animals in this study were examined clinically to determine the suspected infected animals and then subjected to the collection of epithelial tissue samples. Clinically, the infected buffaloes showed different signs, such as fever, reduced appetite, ulcers in the mouth and foot, and impaired mobility. This study used (Semi-Nested RT-PCR) to detect the SAT2 serotype in buffaloes. The findings indicated that 61 out of 70 local isolates (87.14 percent) tested positive for SAT2. These isolates were subsequently submitted to GenBank and assigned accession numbers ranging from (OR576814.1 - OR576820.1). As the first recorded Iraqi SAT2 isolate. The Iraqi isolates were clustered in the same branch as the Egyptian isolate (MZ097480.1). This study is the first to implement the semi-nested RT-PCR technique for identifying the SAT2 serotype in buffaloes in Iraq. The findings of this study have the potential to provide valuable insights into the formulation and implementation of FMD protection programmers in Iraq.