We describe the molecular identification of human tuberculosis (TB) from vertebral bone tissue samples from three different populations of ancient Egypt. The specimens were obtained from the predynastic to early dynastic necropolis of Abydos (7 individuals, c. 3500–2650 B.C.), from a Middle Kingdom to Second Intermediate Period tomb of the necropolis of Thebes-West (37, c. 2100–1550 B.C.) and from five further Theban tombs used in the New Kingdom and the Late Period (39, c. 1450–500 B.C.). A total of 18 cases tested positive for the presence of ancient DNA (aDNA) of the M. tuberculosis complex. Out of the 9 cases with typical macromorphological signs of tuberculous spondylitis, 6 were positive for mycobacterial aDNA (66·7%). Of 24 cases with non-specific pathological alterations, 5 provided a positive result (20·8%). In 50 cases of normally appearing vertebral bones 7 tested positive (14·0%). There were only minor differences in the frequencies between the three populations. These data strongly support the notion that tuberculosis was present and prevalent in ancient Egypt since very early periods of this civilization. The unexpectedly high rate of mycobacterial aDNA in normal bone samples is presumably due to a pre- to perimortal systemic spread of the bacteria and indicates a generalized infection by M. tuberculosis.