Restriction fragment length polymorphism (RFLP) analysis of IS6110 is commonly used to DNA fingerprint Mycobacterium tuberculosis. However, low-copy (< or =5) IS6110 M. tuberculosis strains are poorly differentiated, requiring secondary typing. When spoligotyping was used as the secondary method, only 13% of Maryland culture-positive tuberculosis (TB) patients with low-copy IS6110-spoligotyped clustered strains had epidemiologic linkages to another patient, compared to 48% of those with high-copy strains clustered by IS6110 alone (P < 0.01). Spoligotyping did not improve a population-based molecular epidemiologic study of recent TB transmission.