Background: Multidrug resistant (MDR) Acinetobacter species reports have sharply increased from India. The study aimed to characterize all MDR Acinetobacter species and to evaluate the pattern of antibiotic susceptibility from the various clinical samples. Method: This prospective prevalence study conducted from January 2021 to June 2022 in the Department of Microbiologyat our tertiary care teaching institute of central India isolated MDR Acinetobacter speciesfrom various clinical samples using a standard set ofbiochemical tests and antimicrobialsusceptibility testing performed as per Clinical Laboratory Standards Institute (CLSI) guidelines, (M100; 32th Edition).All colistin resistant isolates(by broth microdilution) were screened for plasmid mediated , mobile colistin resistant, mcr-1, mcr-2 and mcr-3 gene. Results: A total 658 Acinetobacter species could be cultivated from 21,019-culture positive clinical specimens accounting for an isolationrate of 3.13% .Of these, 292 (44.3%) isolates wereMDRAcinetobacter. Out of 292 MDR Acinetobacter species, the most common were Acinetobacter baumannii complex (59.58%), followed by Acinetobacter lwoffii(28.76%) and Acinetobacter hemolyticus(11.64%). Majority of MDR Acinetobacter baumannii complex were from specimen blood (69.47%), followed by pus (56.52%). Around 4% of MDR Acinetobacter species were resistant to colistin. All 6 colistin resistant strains were found to be negative for plasmid mediated mcr-1,2 and 3 genes. Conclusion: Our study showed a high prevalence (44.3%) of MDRAcinetobacter species. The emergence of multidrug resistant Acinetobacter species has become a global public health concern, and ultimately one health concern with a potential to cause serious nosocomial infections. MDR Acinetobacter species were 54.5% and 38.2% resistant to carbapenems and aminoglycosides respectively. Early detection is necessary for optimizing antibiotic use and implementation of strict infection control practices to narrow down the development of antimicrobial resistance.