We have developed a real-time genotyping and quantitative PCR (RT-GQ-PCR) assay to genotype cytomegalovirus (CMV) and quantify viral loads simultaneously in solid organ transplant (SOT) recipients. Special minor-groove DNA-binding probes were designed based on sequence polymorphism in the gB gene to increase genotyping specificity for gB1 to gB4. For validation, 28 samples with known genotypes determined by restriction fragment analysis (RFA) and 121 with unknown genotypes were tested. All samples were from SOT patients with CMV viremia. A 100% concordance for genotyping was achieved by using the RT-GQ-PCR with known genotypes determined by RFA. The RT-GQ-PCR identified more cases of CMV infections with mixed genotypes than RFA did. No cross-reaction between genotypes was observed. All four gB genotypes were detected in the 121 samples of unknown genotype. gB1 was the predominant single genotype (n = 61, 50.4%), followed by gB2 (n = 26, 21.0%), gB3, (n = 11, 9.1%), and gB4 (n = 3, 2.5%). Mixed-genotype infections were detected in 17% (20/121) of the samples. Patients with mixed-genotype infections had significantly higher CMV viral loads than those with single-genotype infections (P = 0.019). The RT-GQ-PCR assay was found to be highly sensitive and specific, with a wide dynamic range (2.7 to 10.7 log(10) copies/ml) and very good precision (coefficient of variation, approximately 1.78%). With the prominent feature of concurrent CMV gB genotyping and quantitation in a single reaction, the new assay provides a rapid and cost-effective method for monitoring CMV infection in SOT recipients.