Tuberculosis, caused by Mycobacterium Tuberculosis is a major public health problem. India is the highest tuberculosis burden country across the globe. Under the Revised National Tuberculosis Control Programme (RNTCP) diagnosis of pulmonary tuberculosis is done by sputum smear microscopy using Zeil-Nelson staining method. For sputum microscopy, two sputum (spot and early morning) samples are collected. Spot sample is collected at the time of first visit of patient to laboratory and early morning sputum is collected the next day. This study is based on retrospective analysis of spot and early morning sputum microscopy samples tested in the year 2012 and 2013 at Designated Microscopy Center (DMC) of Government Medical College, Vadodara, Gujarat. This study showed that the observed difference in sputum sample positivity in the two groups, viz. spot sample and morning sample in diagnosis of TB is statistically insignificant (Chi-square value 1.703 and 0.00584 respectively, P value 0.191 and 0.939 respectively).