A cross-sectional study was carried out on 400 equines holdings (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 ELISA as a screening test and PCR-RFLP targeting Beta giardin loci. In addition, PCR targeting triose phosphate isomerase gene (tpi) specific for assemblage A and B were used as confirmatory. 34 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% CI; 4.8-10.1). Of the 30 positive animals/holdings, 18, 4 and 8 had assemblage A, B and E. Assemblage A was significantly (p < 0.05) more prevalent when compared to assemblages B and E. The total infection rates of Giardia, assemblage B and E were significantly (p < 0.05, Chi-square) higher in donkeys 14.8%, 2.7%, 5.5% compared to horses 5.8%, 0.6%, 1.2%, respectively. Analysis of risk factors revealed that only season was significantly associated with the different Giardia assemblages. Autumn (OR = 0.09) was associated with Giardia infection regardless of the assemblage type as reducing factor. The odds of infection of assemblage 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.