We showed earlier that human beta-thymosin 15 (Tb15) is up-regulated in prostate cancer, confirming studies from others that propagated Tb15 as a prostate cancer biomarker. In this first report on mouse Tb15, we show that, unlike in humans, four Tb15-like isoforms are present in mouse. We used phylogenetic analysis of deuterostome beta-thymosins to show that these four new isoforms cluster within the vertebrate Tb15-clade. Intriguingly, one of these mouse beta-thymosins, Tb15r, consists of two beta-thymosin domains. The existence of such a repeat beta-thymosin is so far unique in vertebrates, though common in lower eukaryotes. Biochemical data indicate that Tb15r potently sequesters actin. In a cellular context, Tb15r behaves as a bona fide beta-thymosin, lowering central stress fibre content. We reveal that a complex genomic organization underlies Tb15r expression: Tb15r results from read-through transcription and alternative splicing of two tandem duplicated mouse Tb15 genes. Transcript profiling of all mouse beta-thymosin isoforms (Tb15s, Tb4 and Tb10) reveals that two isoform switches occur between embryonic and adult tissues, and indicates Tb15r as the major mouse Tb15 isoform in adult cells. Tb15r is present also in mouse prostate cancer cell lines. This insight into the mouse Tb15 family is fundamental for future studies on Tb15 in mouse (prostate) cancer models.