The interferon-activatable Ifi200 gene cluster is located on mouse Chromosome 1q21-q23. We report here our analysis of two genomic regions encoding at least 10 closely related 200 family genes (Ifi201, Ifi202a, Ifi202b, Ifi202c, Ifi203a, Ifi203b, Ifi203c, Ifi203', Ifi204, and Ifi204') in 129/SvJ mice. Through a BAC-based sequencing approach, the exact structure and organization of these highly similar Ifi200 genes were obtained. A high degree of conservation (99% identity) was observed between Ifi202a and b and between Ifi203a and b. The presence of an additional transcribed region in intron 4 of Ifi203a and b suggests the possibility of alternative splicing, and a spliced variant of the Ifi204' mRNA exhibits 91% sequence identity with a related but unmapped D3 mRNA. Comparative analysis of the mouse and human clusters indicates an absence of significant sequence conservation in noncoding sequences, suggesting that the 200 family emerged prior to human-mouse speciation and subsequently diverged after gene duplication.