Initial multidrug-resistant (MDR) tuberculosis (TB) in HIV-negative patients treated at a Buenos Aires referral hospital from 1991 to 2000 was examined by using molecular clustering of available isolates. Of 291 HIV-negative MDRTB patients, 79 were initially MDR. We observed an ascending trend of initial MDRTB during this decade (p=0.0033). The M strain, which was responsible for an institutional AIDS-associated outbreak that peaked in 1995 to 1997, caused 24 of the 49 initial MDRTB cases available for restriction fragment length polymorphism. Of those, 21 were diagnosed in 1997 or later. Hospital exposure increased the risk of acquiring M strain-associated MDRTB by approximately two and a half times. The emergence of initial MDRTB among HIV-negative patients after 1997 was apparently a sequel of the AIDS-related outbreak. Because the prevalence of M strain-associated disease in the study population did not level out by the end of the decade, further expansion of this disease is possible.