Recent phylogenetic studies revealed close relationships between several families of Heterotremata crabs. In this context, we describe the spermatozoal ultrastructure in several Aethridae, Menippidae, Calappidae, Parthenopidae, Cancridae, and Leucosiidae species to elucidate the evolution of spermatozoal characters. The spherical spermatophore in all Heterotremata studied here have a clear wall or pellicle. Spermatozoal results indicate that the fingerprint-like acrosome ray zone is a synapomorphy among these closely related families, including Menippidae, while the parallel acrosome ray zone is an autapomorphy occurring in Portunidae. The striations in the subopercular material are also a synapomorphic character for all studied families while absence is a homoplastic trait and apomorphic to Parthenopidae and Cancridae. Moreover, our results indicate a sharing of certain spermatozoal traits between Aethridae and Portunidae and in the Menippidae Menippe nodifrons. In Cancridae and Parthenopidae, the perforate operculum is a homoplastic character while the perforatorial chamber penetrating the operculum is the main synapomorphy of Cancridae. In Calappidae and Portunidae, the absence of the inner acrosome zone is an apomorphy. The presence of a broad thin, three-layered, operculum filled with a granular matrix is a synapomorphy of the Parthenopidae. Finally, in Leucosiidae, the inner acrosome zone positioned at the mid-point of the acrosome vesicle and the presence of a peculiar type of periopercular rim are a synapomorphy of the group. Overall, our ultrastructural findings align with recent phylogenetic analyses conducted within the Heterotremata clade, providing complementary support and reinforcing the value of spermatozoal ultrastructure as a tool in phylogenetic studies, as it demonstrates clear potential for resolving taxonomic issues.