The shell microstructure of 30 species belonging to 14 genera and nine families of protobranchs were newly described using scanning electron microscopy. As a result, 13 microstructures were recognized. The shell microstructural composition of protobranchia is conservative at a superfamily level among the Recent species, whereas this trend is not necessarily apply to that of fossil protobranchs. Greater diversity of the shell microstructure was revealed in protobranchs than previously known. (1) Nuculoidea commonly have outer prismatic and middle/inner nacreous structures. The outer prismatic layer is newly classified into five types in Nuculidae. In particular, the irregular fibrous prismatic structure was first observed in Nuculoidea. The interspecific variability of outer prismatic layers seems to provide crucial criteria for phylogenetic grouping in this superfamily. (2) Nuculanoidea have non-nacreous structures including the homogeneous, fibrous prismatic and fine complex crossed lamellar structures. Sareptidae, which should presumably be reclassified from Nuculoidea to Nuculanoidea also follows this pattern. (3) Manzanelloidea have microstructural components similar to those of Nuculanoidea. These features strongly suggest the shell microstructure of protobranchs provide useful bases for higher systematics.