Diagnosis of pulmonary tuberculosis (TB) relies on a sputum sample, which cannot be obtained from all symptomatic individuals. Mycobacterium tuberculosis (Mtb) transrenal DNA (trDNA) has been detected in urine, an easily obtainable, noninvasive, alternative sample type. However, reported sensitivities have been variable and likely depend on collection and assay procedures and aspects of trDNA biology. We analyzed three serial urine samples from each of 75 adults with culture-confirmed pulmonary TB disease in Lima, Peru for detection of trDNA using short-fragment real-time PCR. Additionally, we examined host, urine, and sampling factors associated with detection. Overall per-sample sensitivity was 38 % (95 % Confidence Interval [CI] 30–45 %). On an individual level (i.e., any of the three samples positive), sensitivity was 73 % (95 % CI: 62–83 %). Sensitivity was highest among samples from patients with smear-positive TB, 92 % (95 % CI: 62–100 %). Specificity from a single sample from each of 10 healthy controls was 100 % (95 % CI: 69–100 %). Adjusting our assay positivity threshold increased individual-level sensitivity to 88 % (95 % CI: 78–94 %) overall without affecting the specificity. We did not find associations between Mtb trDNA detection and individual characteristics or urine sample characteristics. Overall, our results support the potential of trDNA detection for TB diagnosis.