MicroRNAs (miRNAs) form an abundant class of non-coding RNA genes that have an important function in post-transcriptional gene regulation and in particular modulate the expression of developmentally important transcription factors including Hox genes. Two families of microRNAs are genomically located in intergenic regions in the Hox clusters of vertebrates. Here we describe their evolution in detail. We show that the micro RNAs closely follow the patterns of protein evolution in the Hox clusters, which is characterized by cluster duplications followed by differential gene loss. J. Exp. Zool. (Mol. Dev. Evol.) 304B: 1–10, 2005. © 2005 Wiley-Liss, Inc.