Palaemonid shrimps form a large and diversified group distributed primarily in marine waters. Within the family Palaemonidae the subfamily Palaemoninae is composed of 26 genera and more than 370 species inhabiting marine and fresh waters in epigean and hypogean habitats. One of the relevant questions that have emerged in relation to the subfamily is how the invasion to freshwater has occurred in Mexico, where six freshwater genera belonging to the Palaemoninae (Creaseria, Cryphiops, Macrobrachium, Palaemonetes, Neopalaemon and Troglomexicanus), are present. The purpose of this study was to explore the relationships among these freshwater lineages through a phylogenetic analysis based on partial sequences of the 16S mitochondrial gene to elucidate if these genera are closely related and derived from a common ancestor or if each lineage has invaded the freshwater habitat independently. The analysis included species representing monotypic and diversified genera, from epigean and hypogean habitats and with complete and abbreviated larval development. The results suggest that the invasion of freshwater occurred on four different occasions and that the different lineages originated before the invasions of freshwater occurred.