The evolutionary dynamics of the tetra-nucleotide microsatellite locus Spl-106 were investigated at the repeat and flanking sequences in 137 individuals of 15 Acipenseriform species, giving 93 homologous sequences, which were detected in 11 out of 15 species. Twenty-three haplotypes of flanking sequences and three distinct types of repeats, type I, type II and type III, were found within these 93 sequences. The MS-Align phylogenetic method, newly applied to microsatellite sequences, permitted us to understand the repeat and flanking sequence evolution of Spl-106 locus. The flanking region of locus Spl-106 was highly conserved among the species of genera Acipenser, Huso and Scaphirhynchus, which diverged about 150 million years ago (Mya). The rate of flanking sequence divergence at the microsatellite locus Spl-106 in sturgeons is between 0.011% and 0.079% with an average at 0.028% per million years. Sequence alignment and phylogenetic trees produced by MS-Align showed that both the flanking and repeat regions can cluster the alleles of different species into Pacific and Atlantic lineages. Our results show a synchronous evolutionary pattern between the flanking and repeat regions. Moreover, the coexistence of different repeat types in the same species, even in the same individual, is probably due to two duplication events encompassing the locus Spl-106 that occurred during the divergence of Pacific lineage. The first occured before the diversification of Pacific species (121-96 Mya) and led to repeat types I and II. The second occurred more recently, just before the speciation of A. sinensis and A. dabryanus (69-10 Mya), and led to repeat type III. Sequences in the same species with different repeat types probably corresponds to paralogous loci. This study sheds a new light on the evolutionary mechanisms that shape the complex microsatellite loci involving different repeat types.