Giant clams are conspicuous bivalves inhabiting Indo-Pacific reefs. Since Rosewater’s seminal paper in 1965, the number of giant clam species described or resurrected has exactly doubled. The increased number of species reported and accessibility to genetic material of rare or uncommon species therefore call for a reappraisal of the phylogenetic relationships within the Tridacninae subfamily. Here, we aim to reconstruct the evolutionary relationships among all 12 extant species by performing a comprehensive phylogenetic analysis of mitochondrial genome and nuclear 18S rRNA data from a combination of genome skimming, Sanger sequencing and previously published sequences. Comparing the mitogenomes among Tridacninae species, we report two new findings: (1) the T. crocea mitogenome length obtained here (18,266 bp) is shorter than previously known, and (2) the mitochondrial gene order in T. crocea and T. squamosa differs from the other species. Our phylogeny based on a concatenated 16-gene dataset (15 mitochondrial markers and nuclear 18S rRNA) reveals highly supported relationships within and between the three subgenera, Tridacna (Tridacna), Tridacna (Persikima) and Tridacna (Chametrachea). Overall, the inclusion of new molecular markers greatly improves the confidence and support for the subfamily’s phylogeny. The availability of this comprehensive phylogenetic dataset serves as the foremost baseline of Tridacninae relationships to support future studies examining giant clam systematics, ecology and conservation.