A cross-sectional study was carried out on 400 equine holding (326 horses and 74 donkeys) samples to determine the prevalence of Giardia assemblages A, B, and E in Jordan. Identifying the Giardia assemblages was carried out using enzyme-linked immunosorbent assay (ELISA) as a screening test and PCR-RFLP targeting β-giardin loci. In addition, polymerase chain reaction targeting triose phosphate isomerase gene specific for assemblages A and B were used as confirmatory. Thirty-four samples tested positive by ELISA for Giardia with an apparent prevalence of 8.5%. The PCR-RFLP test confirmed Giardia assemblages in 30 of the 34 ELISA-positive samples giving a true prevalence of 7.7% (95% confidence interval: 4.8–10.1). Of the 30 positive animals/holdings, 18, 4, and 8 had assemblages A, B, and E. Assemblage A was significantly (P < .05) more prevalent when compared to assemblages B and E. The total infection rates of Giardia, assemblages B and E were significantly (P < .05, chi-square) higher in donkeys 14.8%, 2.7%, and 5.5% compared to horses 5.8%, 0.6%, and 1.2%, respectively. Analysis of risk factors revealed that only season was significantly associated with the different Giardia assemblages. Autumn (odds ratio [OR] = 0.09) was associated with Giardia infection regardless of the assemblage type as reducing factor. The odds of infection of assemblages A and E increased in winter (OR = 6.8) and spring (OR = 4.5), respectively. Giardia assemblages A, B, and E infect both horses and donkeys in Jordan with potential impact on human and animal health, and the odds of infections is significantly associated with season.