Mannheimia haemolytica (M. haemolytica) causes shipping fever and several other respiratory diseases in bovine but its involvement in diseases of Gallus gallus domesticus is limited to secondary infections with limited data available from Pakistan. Samples from layer hens showing respiratory clinical signs were processed for isolation and identification of causative agent through biochemical and molecular tests and the isolates were further tested for pathogenicity index. Phylogenetic studies were also done to establish the phylogeny of circulating species. Our study led to the isolation of a Gram-negative, non-motile, coccobacillus from the clinical samples and was identified as M. haemolytica which showed 99% homology with the M. haemolytica serotype A2 of bovine origin upon sequencing of its 16S rDNA gene. A single, smooth colony of this bacterium inoculated in five, 7-day-old embryonated eggs via the yolk sac route induced hemorrhages and 100% mortality. These results were further supported by the recovery of bacterium M. haemolytica from 8 week-old layer hens showing mild clinical signs within 1-week post challenge with the isolate. Our study provides evidence that M. haemolytica may cause clinical disease in Gallus gallus domesticus under stressful conditions and adds to the existing knowledge for differential diagnosis of respiratory pathogens in poultry.