Background: Gram-positive bacteria are common causes of both community acquired and nosocomial infections. This is compounded by the development of multi-drug resistance in this group of bacteria. The aim of this study is to characterize and evaluate drug susceptibility profile of Gram-positive cocci isolated from different clinical samples in the study area. Objective: The main objective of this study was to determine the prevalence of gram positive cocci and multidrug resistance pattern of Gram positive cocci isolated from different clinical samples. Methods: A laboratory based cross-sectional study was conducted to determine the prevalence of multidrug resistant strains of Gram-positive bacteria isolated from 792 patients referred to Arsho Advanced Medical Laboratory, from January to April 2018.Different clinical samples were collected and inoculated onto primary isolation culture media, blood culture bottles and blood agar. Preliminary identification was carried on based on number, type, morphology appearance of colonies and Gram reaction after growth on culture media. Identification and drug susceptibility testing also carried out by GP and AST-GP71 cards of the VITEK 2 compact systems