Members of the Mycobacterium avium complex are distinguished by the presence of highly antigenic surface molecules called glycopeptidolipids (GPLs) and the oligosaccharide portion of the serovar-specific GPL defines the 28 serovars. Previously, the genomic region (ser2) encoding the enzymes responsible for the glycosylation of the lipopeptide core to generate the serovar-2-specific GPLs has been described. In this work, the ser2 gene clusters of M. avium serovar 2 strains 2151 and TMC 724 were fully sequenced and compared to the homologous regions of M. avium serovar 1 strain 104, M. avium subsp. paratuberculosis and M. avium subsp. silvaticum. It was also determined that 104Rg, a mutant of strain 104 that produces truncated GPLs, lost several GPL biosynthesis genes by deletion. This comparison, together with analysis of protein similarities, supports a biosynthetic model in which serovar-2-specific GPLs are synthesized from a serovar-1-specific GPL intermediate that is derived from a non-specific GPL precursor. We also identified a gene encoding an enzyme that is necessary for the biosynthesis of serovar-3- and 9-specific GPLs, but not serovar-2-specific GPLs, suggesting that the different serovars may have evolved from the acquisition or loss of genetic information. In addition, a subcluster of genes for the biosynthesis and transfer of fucose, which are needed to make serovar-specific GPLs such as those of serovar 2, is found in the non-GPL-producing M. avium subspecies paratuberculosis and silvaticum.