Construction materials from the internal ducts of Aqua Traiana, a still operative Roman aqueduct built in 109 AD to supply water to Rome, were characterized by optical microscopy (OM), scanning electron microscopy (SEM-EDS), X-ray powder diffraction (XRPD) and electron microprobe analysis (EMPA). Petrographic analysis and XRPD revealed that mortar aggregates are compatible with Vitruvius’ harena fossicia and allowed the distinction of the original mortars from those of the 17th-century papal restoration. The first showed an amorphous binder while the latter have a typical lime binder. By SEM-EDS and EMPA, the microstructure of mortar aggregates was analyzed and the composition of specific minerals quantified. Microanalysis testifies the Romans’ great expertise in the selection of pozzolanic building materials, giving evidence of the possible use of local tuffs from the Sabatini Volcanic District. It also confirms the exploitation of red pozzolan from the Roman Magmatic Province, specifically from the Alban Hills district. OM also proves a high compatibility with local supplies for bricks and cocciopesto. Of these, the first were fired at moderately low temperature, while the latter show an amorphous binder as in the original Trajan mortars. All building materials thus stand for similar technological choices and a coeval production.