Drosophila frequenin (Frq), the homolog of the mammalian neuronal calcium sensor-1 (NCS-1), is a high affinity calcium-binding protein with ubiquitous expression in the nervous system. This protein has an important role in the regulation of neurotransmitter release per synapse, axonal growth and bouton formation. In D. melanogaster, frequenin is encoded by two genes (frq1 and frq2), a very unexpected feature in the Frq/NCS-1 subfamily. These genes are located in tandem in the same genomic region, and their products are 95% identical in their amino acid sequence, clearly indicating their recent origin by gene duplication. Here, we have investigated the factors involved in this unusual feature by examining the molecular evolution of the two frq genes in Drosophila and the evolutionary dynamics of NCS family in a large set of bilaterian species.Surprisingly, we have found no amino acid replacements fixed across the twelve Drosophila species surveyed. In contrast, synonymous substitutions have been prevalent in the evolution of the coding region of frq1 and frq2, indicating the presence of strong functional constraints following gene duplication. Despite that, we have detected that significant evolutionary rate acceleration had occurred in Frq1 in early times from the duplication, in which positive selection (likely promoting functional diversification) had probably an important role. The analysis of sequence conservation and DNA topology at the non-coding regions of both genes has allowed the identification of DNA regions candidates to be cis-regulatory elements. The results reveal a possible mechanism of regulatory diversification between frq1 and frq2.The presence of two frequenins in Drosophila and the rapid accumulation of amino acid substitutions after gene duplication are very unusual features in the evolution of the Frq/NCS-1 subfamily. Here we show that the action of positive selection in concordance with some extent of regulatory diversification might explain these findings. Selected amino acid substitutions in Frq1 likely contributed to the functional divergence between the two duplicates, which, in turn, should have diverged in their regulation by ecdysone-induced early genes.