Background: Technetium-99m radioisotope is the backbone of any nuclear medicine institute. This radioisotope is acquired by the elution of Molybdenum-99/Technetium-99m generator which emits 141 keV gamma ray and having 6.04 hour half-life. Occasionally the contents of Molybdenum-99 may mix up with elute, which may affect image quality and patients may receive higher doses than recommended. This study was initiated to check and evaluate the contamination of Molybdenum-99 in Molybdenum-99/Technetium-99m generators used at Nuclear Institute of Medicine and Radiotherapy (NIMRA) Jamshoro Pakistan before its administration to patients for diagnostic procedures. Materials and Methods: The Molybdenum-99 impurity in Technetium-99m elute for 50 generators was studied during the year 2012. The measurements were made using dose calibrator and a standard canister at the time of first elution of the generators. The dose calibrator displays result after executing various steps in a sequence. Due to some production process error or any mechanical fault high activity of Molybdenum-99 was recorded and removed with decrease in Technetium-99m yield. Results: The most of eluted generators (90%) contained 10% of molybdenum breakthrough of the permissible limit (P < 0.05). The high activity of Molybdenum-99 was recorded in only 6% of the generator and removed by using standard methods with some reduced Technetium-99m activity. Conclusion: The molybdenum breakthrough was according to recommended standards in most of the generators. The removal of high contents of Molybdenum-99 enabled the institute to continue routine imaging services by improved image quality and reduction in radiation dose to the patients.