Giardia lamblia, one of the most common intestinal protozoan pathogens worldwide, causes human gastrointestinal diseases. Because of its high prevalence and major impact on the patients’ quality of life, giardiasis has become one of the most important public health problems. We aimed to evaluate the prevalence and assemblages of G. lamblia isolated from referred individuals to the medical laboratories in the center of Kurdistan province, West of Iran. Direct wet mount and formalin-ether sedimentation technique were applied for microscopy determination of Giardia infection in 1383 obtained stool samples. DNA was extracted from positive stool samples and amplified by Nested-PCR using specific primers of G. lamblia Trios phosphate isomerase (TPI) gene. Genotyping and assemblages was determined using sequence analysis of the TPI gene. Based on microscopic examination, out of the 1383 samples, 23 (1.66%) G. lamblia were detected. Genotyping data indicated 12 (52%) cases as assemblage a cluster, and 11 (48%) cases as assemblage B cluster. Molecular analysis in this study reveal that assemblages A and B are the most common types of human giardiasis in Kurdistan province and giardiasis could be a zoonosis disease. Molecular identification of Giardia isolates from animals and control programs is recommended to prevent the spreading of giardiasis in humans.