Glycoprotein B (gB) of human cytomegalovirus (CMV) is the major target protein of neutralizing antibodies, and four variant types are known. A previously reported polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method was modified by eliminating the cell culture step and amplifying a CMV fragment (corresponding to a.a. 441-450 of gB) by nested PCR. DNA was extracted from the sera of 27 pediatric bone marrow transplant recipients and subjected to nested PCR. Of the samples, 20 yielded PCR products, and the gB type was determined by RFLP. Of the 20 patients, 4 (20%) had gB type 1 (Towne type), 15 patients (75%) had gB type 2 (AD 169 type), and 1 patient (5%) had gB type 3. Previous studies showed that gB type was most common among Caucasians [Chou and Dennison: J Infect Dis 1991;163:1229-1234; Fries et al: J Infect Dis 1994;169:769-774]. Thus, gB genotypes seem to be distributed differently in Caucasians and Japanese. Further, nucleotide sequence analysis of the amplified region revealed that all the type 2 viruses had the same amino acid sequences. The type 3 sample had a novel amino acid substitution at position 498. Of the type 1 samples, 3 had amino acid substitutions in various positions: 1 sample at position 493,1 sample at position 447 and 1 sample at position 452, 493 and 498.