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Background: Distribution of Tuberculosis (TB) has a special template affected by geographic and social factors. Nowadays Geographical Information System (GIS) software is one of the most important and useful epidemiologic devise for identifying geographic areas and high risk population for infection by TB. Aim of this study was assessment of Tuberculosis distribution by GIS in Khuzestan province, Iran. Methods: In this retrospective cohort study, documents of 6363 TB patients identified in Khuzestan province at the beginning of 2005 until the end of the September of 2012 were assessed and sampling method was census. The data was gathered by TB-Register Center. TB incidence was evaluated by GIS maps according to climatic and vegetation with 1:25,000 scale. The data was analyzed in SPSS software and means plot and Independent's t-test, ANOVA with a significance level of less than 5% and Eta Correlation. Results: Spread of Tuberculosis in climatic and vegetation were plotted in GIS maps, cumulative incidence rate was calculated separately for each area. There was a statistical significant relationship between cumulative incidence rate of disease and climatic (P=0.017) and the Eta correlation coefficient was 0.379 (P=0.011). There was a statistical significant relationship between cumulative incidence rate of TB and vegetation (P=0.004) and the Eta correlation coefficient was 0.388 (P=0.188). Mean age and median age of patients were 39.3±18 and 35±6.5 years respectively. For women, mean age was 2.1 years older than men and this difference was statistically significant. (P=0.0001). Conclusion: The risk of TB infection is closely associated with the climatic and vegetation. So the risk of TB in extra-dry climate and vegetation of highland meadows is more than other areas. Special care and services to high-risk areas is recommended. [ABSTRACT FROM AUTHOR]