A high-resolution melting (HRM) assay was applied to distinguish between Giardia duodenalis assemblages A and B from human and dog feces based on the triosephosphate isomerase gene (tpi). The genomic DNAs were selected from assemblages A (WB) and B (GS) as reference and plasmids were constructed. The reference plasmids and genomic DNAs from 15 Giardia-positive samples were analyzed by HRM assay. This was followed by separate real-time PCR assays specific for assemblages A and B using EvaGreen (EG) to identify PCR products by melting-point analysis. Our results indicate that PCR with HRM in a one-step closed-tube method is a reliable diagnostic method for G. duodenalis zoonotic assemblage identification and more rapid than restriction length polymorphism analysis and direct sequence analysis, HRM is specific, sensitive, reproducible, and rapid. This study is the first use of EG dye for Giardia genotyping. This assay is a promising approach to determine the presence and genotype of Giardia based on a highly variable gene.