Rationale In 2002 a mobile radiographic screening program was started in Rotterdam to respond to high rates of tuberculosis among illicit drug users and homeless persons. Objectives We studied trends and characteristics of tuberculosis among these risk groups and assessed the impact of the screening program on transmission using molecular typing. Methods Description of trends, demographic and disease-related characteristics of tuberculosis cases among these risk groups between 1993 and 2005. Tuberculosis was considered resulting from recent transmission if the mycobacterial DNA fingerprints of cases were identical with those of other cases in the risk groups in the previous two years. Results During the study period, 206 tuberculosis cases among illicit drug users and homeless persons were notified, representing 11.4% of the total case-load of 1,807 in Rotterdam. The annual number of tuberculosis cases declined from 24 at the start of the screening program to 11 cases in 2005. The screening program identified 28 cases (a prevalence rate of 327 per 100,000 radiographs) of which 12 were smear-positive. In the years 1997-2002 more than 80% of the illicit drug users or homeless persons with tuberculosis was infected with one of the Mycobacterium tuberculosis strains prevalent among these risk groups. After nearly four years of systematic radiographic screening this proportion declined to 45% in 2005. Conclusions Radiographic screening among illicit drug users and homeless persons decreased incident cases and reduced disease transmission in Rotterdam. DNA fingerprinting can be a useful tool to evaluate the impact of a tuberculosis screening program. Such screening of illicit drug should be continued to prevent a resurgence of tuberculosis.