Background: Information on strain types of human cytomegalovirus (HCMV) isolates from Saudi Arabian patients is lacking. Materials and Methods: 80 clinical isolates of HCMV from Saudi Arabian patients were analyzed by PCR amplification of three regions (DNA polymerase, glycoprotein B, and glycoprotein H) of the virus genome. The resultant amplicons (2.0-2.7 kb) were further studied by restriction fragment Length polymorphism (RFLP) using four enzymes (HaeIII, HhaI, MspI, and RsaI). Results: Combined analysis of the cleavage patterns generated by the enzymes identified five strains, S1-S5, and several mixed and unique strains. 18 isolates belonged to S1 strain and were similar to laboratory strain AD169. Eight isolates were present in each of S2 and S3 strains. Six isolates and four isolates were found in S4 and S5 strains, respectively. 12 isolates contained a mixture of S3 and S5, which may have resulted from a dual infection. Each of the 24 remaining isolates had a different strain pattern. Conclusion: Our findings show that 80 HCMV clinical isolates were distributed into 30 different strains using PCR-RFLP analysis of multiple viral subgenomic regions. However, the number of isolates is not uniformly distributed among strains (p < 0.02).