Analysis of nucleotide sequence variation at a microsatellite DNA locus revealed extensive size homoplasy of alleles in Ad鬩e penguins (Pygoscelis adeliae). Variation in the flanking regions at this locus allowed discrimination between mechanisms proposed for length changes in microsatellite DNA alleles. We further examined the structure of alleles for the same microsatellite DNA locus across 11 additional species of penguin (Spheniscidae) by mapping allele sequences onto an independent penguin phylogeny. Our analysis indicated that the repeat motifs appear to have evolved independently on several occasions. We observed sequence instability in the region bordering the repeat tract with a transversional bias predominating. We propose that this bias results from inaccurate DNA replication owing to the sequence context of this repeat tract. Because we show that regions flanking repeat sequences exhibit this mutational bias, this cautions against the use of such regions for phylogeny reconstruction.