Aspergillus spp. is frequently isolated in respiratory samples from patients with severe chronic obstructive pulmonary disease; however, the clinical significance of this mold is unclear and its presence may indicate temporary passage, benign chronic carriage, or onset of invasive disease. This hospital based cross-sectional observational study was carried out at the Department of TB and Chest, Index Medical College, Indore (M.P.). Detection of Aspergillus species was done using culture and Polymerase Chain Reaction. Sensitivity, specificity, of 10.0% KOH, and culture findings against PCR was found to be 68.7%, 95.1% respectively for KOH and 72.9% and 100.0% respectively for culture test. Species detection in nested PCR was A. niger (2.0%), A.fumigatus (11.3%) and A.flavus (10.0%). This shows more species specificity of PCR. Aspergillusfumigatus was the most dominant molds isolated from sputum in aspergillosis patients. Molecular methods are more sensitive and specific methods. It gives results for detection and speciation in short time which helps early diagnosis and treatment of patient