Background: Delays in the diagnosis of tuberculosis reflect a lack of access to care, and contribute to ongoing tuberculosis transmission in the community. The objective of this study was to evaluate the delay in tuberculosis testing and the associated risk factors in Shanghai, Shandong and Sichuan provinces in China. Methods: A prospective cohort study of 765 culture-positive pulmonary tuberculosis patients registered between December 2006 and December 2008. The delay between the onset of symptoms and tuberculosis diagnosis testing and patient information were recorded in a questionnaire and analysed. Results: The median delay was 36 days and was significantly shorter in patients from Shanghai compared with other places (30 vs. 42 days, P < 0.001). Multivariate analysis revealed that cough in Shanghai patients, lowest income level, being married and presenting expectoration in Shandong and Sichuan patients, were associated with a delay in the diagnosis testing of tuberculosis of >30 days. The only factor associated with a delay of >90 days was, in Shandong and Sichuan provinces only, female gender. The presence of other pulmonary symptoms like haemoptysis and loss of weight, fever and chills could shorten these delays. Conclusion: Efforts to shorten delays in the diagnosis of tuberculosis must target vulnerable populations. The non-specific symptom of cough is a risk factor associated with longer delays. Training for healthcare workers in areas with a high incidence of tuberculosis, where a delayed diagnosis in coughers may enhance tuberculosis transmission in the community, is of paramount importance.