Spiroplasma species (Mollicutes: Spiroplasmataceae) are associated with a wide variety of insects, and serology has classified this genus into 34 groups, 3 with subgroups. The 16S rRNA gene has been used for phylogenetic analysis of spiroplasmas, but this approach is uninformative for group VIII because the serologically distinct subgroups generally have similarity coefficients >0.990. Therefore, we investigated the utility of the 16S-23S rRNA spacer region as a means to differentiate closely related subgroups or strains. We generated intergenic sequences and detailed serological profiles for 8 group VIII Spiroplasma strains. Sequence analyses using Maximum Parsimony, Neighbor Joining, and Maximum Likelihood placed the strains into 2 clades. One clade consisted of strains BARC 2649 and GSU5367. The other clade was divided into clusters containing representatives of the 3 designated group VIII subgroups (EA-1, DF-1, and TAAS-1) and 3 previously unclassified strains. The stability of the positions of the strains in various analytical models and the ability to provide robust support for groupings tentatively supported by serology indicates that the 16S-23S intergenic rDNA sequence will prove useful in intragroup analysis of group VIII spiroplasmas.