On-line bioinformatics tools were used to identify and characterize all paralogs (intragenomic homologs) of muscle myosin regulatory light chains in the human genome. The initial search yielded 22 possible paralogs, but careful examination of supporting data eliminated most of these. Five paralogs were clearly identified with the tissue types (skeletal and cardiac muscles, smooth muscle, non-muscle cytoplasm) in which they are expressed. Sequence comparisons and phylogenetic analysis showed early divergence of a common ancestor of smooth muscle and non-muscle paralogs from a common ancestor of skeletal and cardiac muscle paralogs. An unusual sixth human paralog was very similar to the regulatory light chain of “superfast” myosin, which to date has been found only in cat. Finally, a unique and questionable “precursor lymphocyte” paralog was tentatively identified. Three-dimensional structural models of all seven human paralogs were constructed using the known structure of chicken fast skeletal muscle regulatory light chain as a template.