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Enterococci are well-established causative agents of a number of diseases in humans and animals alike. A total of 1047 isolates (838 presumptive enterococci and 209 non-enterococci), related to poultry environment (faeces/manure, feed, water and air), were evaluated for species diversity, virulence potential, antibiotic resistance and associated threats. Multiplex PCR of 204 presumptive enterococci using genus (16s rDNA)- and species-specific (superoxide dismutase) primer set leads to identification of six Enterococcus spp., i.e. Ent. faecium, Ent. faecalis, Ent. mundtti, Ent. hirae, Ent. gallinarum and Ent. casseliflavus. BOX-PCR analysis of the representative strains confirmed inter-strain variation among species. Although enterococci generally appear to be catalase negative, Ent. faecalis from some faecal, water and air samples showed catalase production. In vitro assays identified poultry environment as a reservoir of MVD and MAR enterococci and non-enterococci. In addition to vancomycin, high-level-ampicillin-, clarithromycin-, erythromycin-, kanamycin- and gentamicin-resistant enterococci and non-enterococci are also indicated. Our results show that virulence potential and resistance vary with the source of isolation. Our studies on MAR and MVD enterococci in environment, especially in air and water, clearly indicate the need for a serious commitment to deal with pathogenic enterococci associated with poultry for preventing transmission of infectious agent to humans.Significance and Impact of the Study: This study demonstrates that poultry environment of Karachi city harbours a diverse reservoir of Enterococcus spp. with multiple antibiotic resistance and virulence determinants. It is expected that the results will help in assessing the impact of multiple antibiotic resistance and virulent enterococci on public health, improvement of farm management practices and preventing their dissemination. Our findings strongly suggest the need for reducing antibiotic usage during poultry production, particularly those that are being used to treat human infections.