Mouse annexin VIII cDNA was characterized by DNA sequencing of expressed sequence tag clones, molecular systematic analysis, and genetic linkage mapping to investigate its evolutionary origin. Its subfamily identity, divergence pattern, and nucleotide substitution rate were established by comparison with other annexin cDNA and deduced protein sequences. The known phylogenetic association of annexin VIII in an evolutionary clade with annexins XI, IV, V, and VIa identified these close homologs as potential progenitors or duplication products. Cladistic analysis confirmed the base position of annexin XI and its relationship to annexin IV as a direct duplication product. Although annexin VIII also derived from annexin XI, the evolutionary branching order, gene separation times, and mapping results indicated that it was probably a subsequent duplication product of annexin IV about 300 million years ago. Dates were calibrated against the assumed separation time of 75 Mya for rodents from other mammals, divergence rates were based on comparisons of all available annexin species, and relative rate tests implied individually stable gene clocks for most annexins. Linkage mapping of mouse Anx8 to the centromeric region of Chromosome (Chr) 14 placed it in a more distal homology group from previously mapped Anx7 and Anx11. Despite their synteny, the combined proximity and segregation of these three annexins diminished the likelihood that they were mutual gene duplication products.