Sputum samples from new tuberculosis (TB) cases were collected over 2 years as part of a prospective study in the northeastern part of Lima, Peru. To measure the contribution of recent transmission to the high rates of multidrug resistance (MDR) in this area, Mycobacterium tuberculosis complex (MTBc) isolates were tested for drug susceptibility to first-line drugs and were genotyped by spoligotyping and 15-locus mycobacterial interspersed repetitive-unit (MIRU-15)-variable-number tandem repeat (VNTR) analysis. MDR was found in 6.8% of 844 isolates, of which 593 (70.3%) were identified as belonging to a known MTBc lineage, whereas 198 isolates (23.5%) could not be assigned to these lineages and 12 (1.4%) represented mixed infections. Lineage 4 accounted for 54.9% (n=463) of the isolates, most of which belonged to the Haarlem family (n=279). MIRU-15 analysis grouped 551/791 isolates (69.7%) in 102 clusters, with sizes ranging from 2 to 46 strains. The overall high clustering rate suggests a high level of recent transmission in this population, especially among younger patients (odds ratio [OR], 1.6; P=0.01). Haarlem strains were more prone to cluster, compared to the other families taken together (OR, 2.0; P<0.0001), while Beijing (OR, 0.6; P=0.006) and LAM (OR, 0.7; P=0.07) strains clustered less. Whereas streptomycin-resistant strains were more commonly found in clusters (OR, 1.8; P=0.03), clustering rates did not differ between MDR and non-MDR strains (OR, 1.8; P=0.1). Furthermore, only 16/51 MDR strains clustered with other MDR strains, suggesting that patients with primary MDR infections acquired the infections mostly from index cases outside the study population, such as retreated cases.