The aim of this study was to establish an assay to compare Mycobacterium tuberculosis strains, and cells grown under different growth conditions, in terms of their ability to cause a lung infection and disseminate to the spleen. M. tuberculosis strains H37Rv, Erdman, South Indian (TMC120, SI) and H37Rv cells grown aerobically or under low oxygen/iron limitation in a chemostat were assayed for infectivity. Groups of 8 animals were challenged with 3 different doses of each strain. Lung and spleen bacteriology was assessed at 16 days post-infection for all strains. Bacteriology and lung pathology at day 56 was studied for H37Rv, Erdman and SI. Strains H37Rv and Erdman had a statistically significantly higher pathogenic potential than SI and this was confirmed by analysis of lung pathology performed at 8 weeks post-infection, although the Erdman strain caused more extensive caseation without calcification and little encapsulation. The model could discriminate between cells grown under different growth conditions; low-oxygen/iron-limited cells had a significantly higher infectivity than those grown aerobically. This study presents a quick and reliable method for comparing with statistical confidence, the pathogenic potential of M. tuberculosis strains and the impact of in vitro growth conditions on the infectivity of M. tuberculosis in vivo.