Background: Conventional and nested polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP) have been used to identify Mycobacterium ulcerans in separate studies and different specimens. However, the sensitivities of these three techniques have not been compared in a single study. Objective: This study compared the performance of two variant PCR techniques and LAMP assays to detect M. Ulcerans in same clinical specimens. Methods: Samples were collected from patients suspected of Buruli ulcer disease (BUD) in Southern Ghana. Ulcerative and non-ulcerative forms of the disease were swabbed and aspirated respectively. Insertion sequence 2404 (IS2404) M. ulcerans targets were detected in each sample using conventional polymerase chain reaction (PCR), nested PCR and loop-mediated isothermal amplification (LAMP) assay. Results: In all, 141 suspected BUD patients were sampled (Amasaman, n = 52; Obom, n = 17; Paakro, n = 31; Nkawie, n = 21 and Tepa, n = 20). The reference technique, nested PCR, detected M. ulcerans in 122 (86.5%) whereas conventional PCR and BU-LAMP detected M. ulcerans in 104 (73.7%) and 119 (84.4%) samples respectively. Compared to nested PCR, conventional PCR performed poorly (x 2 = 19.7, p < 0.01; κ = 0.58; % agreement = 86.62) while BU-LAMP was as good as nested PCR (x 2 = 0.457, p = 0.459; κ = 0.88; % agreement = 97.16). Sensitivity of conventional PCR and BU-LAMP were 84.6% (95% CI: 76.9-90.4) and 97.5% (95% CI: 92.9-99.5) respectively. BU-LAMP and nested PCR detected M. ulcerans in ulcerative forms of the disease and category II lesions better while the three techniques did not differ in sensitivities in other clinical forms and lesion categories. Conclusion: BU-LAMP assay is very comparable to nested PCR in detecting M. ulcerans from clinical specimens. More importantly, LAMP assay is user-friendly, fast, requires less instrumentation and easy to use in resource-limited laboratory with minimal user training.