We compared cetylpyridinium chloride (CPC), ethanol (ETOH) and OMNIgene.SPUTUM (OMNI) for 28-day storage of sputum at ambient temperature before molecular tuberculosis diagnostics.Three sputa were collected from 133 smear-positive TB patients (399 sputa). Each patient's sputum was stored with either CPC, ETOH or OMNI for 28 days at ambient temperature, with subsequent rpoB amplification, targeting a short fragment (81-bp, GeneXpert MTB/RIF (Xpert)) or a long fragment (1764-bp, in-house nested-PCR). For 36 patients, Xpert was also performed at baseline on all 108 fresh sputa.After the 28-day storage (D28), Xpert positivity did not significantly differ between storage methods. In contrast, higher positivity for rpoB nested-PCR was obtained with OMNI (125, 94%), relative to ETOH (114, 85.7%, p=0.001). Smears with scanty AFB had lower rpoB-PCR positivity for ETOH storage (10, 41.7%) relative to CPC (16, 66.7%; difference=25%, 95%CI(3.5-46.5), p=0.031) or OMNI (16, 69.6%; difference=26.1%, 95%CI(3.8-48.4), p=0.031) with no difference between CPC and OMNI. Post-storage Ct-values significantly decreased compared to pre-storage with ETOH (D=-1.1, 95%CI(-1.6 to -0.6), p=0.0001), but not with CPC (p=0.915) or OMNI (p=0.33). For one patient's ETOH- and CPC-stored specimens with a Ct<10, Xpert tested rifampicin false-resistant at D28, which was resolved by repeating Xpert on a 1/100 diluted specimen.In conclusion, 28-day storage of sputum in OMNI, CPC or ETOH at ambient temperature does not impact short fragment PCR (Xpert), including for low smear-grades. However, for long fragment PCR, ETOH yielded a lower PCR positivity for low smear-grades, while the performance of OMNI and CPC was excellent for all smear-grades.