The nucleotide at position 1858 of hepatitis B virus has importance in chronic hepatitis B (HB) because a cytosine at nt 1858 effectively prevents virus escape through the precore TAG stop codon mutation. The relatedness between nt 1858 and genotypes was analyzed using a new genotyping method based on restriction fragment length polymorphism (RFLP) analysis of an S gene amplicon. Seventy-three gene bank sequences were analyzed by phylogenetic tree construction and RFLP prediction. A tree supporting the existence of 6 genotypes (A-F) was obtained, with C-1858 found in 9 of 9 A genotypes, 0 of 7 B, 0 of 19 C, 1 of 10 D, 0 of 2 E, and 3 of 3 F. Serum samples from 187 HB e antigen-positive chronic carriers were analyzed: Genotypes in northern Europeans were 60% A, 31% D; in southern Europeans and Middle Easterners 96% D; in Africans 53% A, 27% D, 20% E; and in East Asians 14% A, 43% B, 43% C. Cytosine at nt 1858 was found in 36 of 44 A, 0 of 32 B, 8 of 34 C, 0 of 59 D, 0 of 3 E, and 1 of 1 F genotype samples.