The mechanism of high altitude effect on tuberculosis (TB) infection has not been fully established. We previously reported a lower positive tuberculin skin test (TST) prevalence in high altitude villages compared with sea level communities in Peru. In this study, four additional communities were tested to assess whether decreased TB transmission was also in urban environments at high altitude. TST results from 3,629 individuals in nine communities were analyzed using generalized estimating equations to account for community clustering. Positive TST prevalence was not significantly different between the urban highland and the urban non-highland communities after adjusting for age, household contacts with a TST-positive person or a TB case, and presence of a Bacillus Calmette-Guérin vaccination scar. The effect of population concentration and increased contact with active TB overwhelmed the protective effect of altitude in urban highlands. Highland cities require the same preventive efforts against TB as non-highland communities.