Aims: This study describes an approach for genotyping Giardia cysts obtained from wastewater treatment plants (WTPs) in Spain using real-time PCR (qPCR) in combination with immunomagnetic beads. Methods and Results: A 50-cycle amplification of a 74-bp fragment of the Giardia beta-giardin gene was adopted from a previous qPCR method. Additionally, two locked nucleic acid (LNA) probes were designed (LNA P434 P1 for assemblage A and LNA P434 H3 for assemblage B). All 16 wastewater samples analysed were positive with the immunofluorescence assay (IFA). Assemblage A was detected in all WTP samples using primer-LNA probe P434 P1 set. Giardia duodenalis identification was confirmed by PCR-RFLP analysis and sequencing of the beta-giardin gene in the water samples found positive by IFA and qPCR. Among the 16 assemblage A isolates that were sequenced, two subtypes were identified; 11 corresponded to the A2 subgenotype, whereas three corresponded to the subgenotype A3. A mixture of subgenotypes was found in the remaining two isolates. Conclusions: The newly developed qPCR assays were able to discern G. duodenalis assemblages A and B in wastewater. Significance and Impact of the Study: The real-time PCR assays provided a rapid method for detection and one-step genotyping of G. duodenalis from wastewater samples, and its application would contribute to understanding the distribution and abundance of G. duodenalis assemblages A and B in wastewater.