The tetranucleotide microsatellite locus Spl-106 has been widely used as a molecular marker in sturgeon studies. To investigate the evolutionary process of this highly variable locus in Acipenseriforms, cross-species amplifications were performed in 130 individuals from 15 species and successful in 13 species. All PCR products were sequenced. According to the flanking sequences, a total of 94 alleles at locus Spl-106 were found in 11 out of 13 species. Twenty-three haplotypic flanking sequences were detected and four of them are dominant types present in 70 out of 94 alleles. Two of the dominant types are species-specific types, and the other two are composed of alleles from species of the Pacific and Atlantic lineages, respectively. The repeat region evolved synchronously with the flanking region. The Atlantic clade was also found in the genealogy tree of the repeat region constructed using the MS Align method. Although the basic repeat structure was variable, several alleles were highly conserved among species and evolved independently. The evolutionary process of this locus in Acipenseriforms was reconstituted from a single repeat (TAGA)n to compound repeats (TAGA)n(TAAA)m, then to another single repeat (TAAA)n, and finally to a totally new compound repeat structure (TAAA)m(GAAA)n. Reciprocally, for the sturgeon phylogeny, our results suggest that Acipenser sturio diverged earlier than Schphirhynchus platorynchus, and infirm the Huso genus, since the two Huso species are classified within the Acipenser species of the Pacific and Atlantic lineages, respectively. Moreover, the sequence information also supports the close relationship between A. sinensis and A. dabryanus, and the relationships among A. transmontanus, A. schrenckii, and H. dauricus.