Colonization of central venous catheters increases the risk of blood stream infections and is associated with increased morbidity, mortality and excess hospital costs. Objectives: To estimate frequency of central venous catheter colonization. Study Design: Cross Sectional study. Setting: Microbiology Laboratory of Punjab Institute of Cardiology, Lahore. Period: May 2018 to December 2018. Material and Methods: Total 116 specimens of central venous catheters were received in the laboratory and processed to isolate various organisms. Organisms were identified on the basis of colonial morphology, Gram staining and biochemical tests. Antimicrobial sensitivity testing was performed and results were recorded. Results: Out of total 116 specimens of central venous catheters, bacterial growth was obtained from 74.1% of central venous catheter tips. From 86 cultures yielding growth, 94 micro-organisms were isolated. The most frequent organisms isolated from central venous catheter tip culture in decreasing order of frequency were Coagulase negative staphylococci (29.8%), S. aureus (24.5%), Candida species (23.4%), Klebsiella species (12.8%), and Acinetobacter spp. (4.3%). Drug susceptibility pattern of isolates was recorded. Conclusion: Central venous catheters can get colonized and lead to catheter related infections. Implementation of prevention programs can result in decrease in colonization rate of catheters and thus Catheter related infections.