Introduction: Bioaerosols are one of major sources of hospital-acquired infections (HAI's) that can pose serious health implications to the patients, health care workers and visitors in the hospitals across the world. Methodology: In this study, the molecular identification and phylogenetic analysis of bioaerosols collected from Orthopedic Wards (OW) and Orthopedic Emergency Rooms (OER) of six hospitals in Lahore, Pakistan was done to investigate their diversity and genetic relatedness. Moreover, the role of different ventilation practices (i.e., centrally air-conditioned and non-central air-conditioned) in determining bioaerosols load was evaluated by using both culture and non-culture based (Flow cytometry) approaches. Results: The molecular characterization based on 16S rRNA gene and phylogenetic analysis of frequently recovered bacterial isolates showed 97-99% similarity to diverse sources i.e., air, soil and clinical strains isolated from various countries. The centrally air-conditioned hospitals had significantly lower levels of bioaerosols at most of the sites as compared to non- central air-conditioned hospitals. Conclusions: The molecular characterization and phylogenetic analysis based on 16S rRNA gene sequences can be effective tool in identifying nature and evolution of bioaerosols, and can improve infection control and surveillance in hospitals. The observed levels of bioaerosols suggest hospitals equipped with central air conditioners have considerably more air hygiene compared to non-central air conditioning systems. These findings are imperative for informing policies on planning and implementation of infection control strategies in hospitals in resource limited settings.