Slovenia is a small Central European country with a population of 1.99 million and an incidence of tuberculosis (TB) of 18.6 per 100,000 inhabitants in 2001. In a prospective nation-wide, 1-y DNA fingerprinting study, the genetic diversity of 99.7% of all Mycobacterium tuberculosis isolates obtained from Slovenian patients with culture-verified TB in 2001 were assessed using a standardized IS6110 restriction fragment length polymorphism (RFLP) method. Among 306 M. tuberculosis isolates, 228 different IS6110 RFLP patterns were found. The number of IS6110 copies varied from 2 to 16 (9.2 copies per isolate on average). Only 2 isolates (0.7%) with less than 5 IS6110 copies were identified. Clustered M. tuberculosis isolates were detected in 116 (37.9%) patients. The degree of recent transmission in the 1-y period was 25%. The clustering rate decreased with age from 46.4% (age group under 35 y) to 19.5% (age group above 65 y). A history of alcohol abuse and homelessness was found to be associated with clustering of TB cases. In conclusion, a high clustering frequency was identified among Slovenian TB patients. The study increased our understanding of important risk factors and routes of TB transmission in Slovenia.