We investigated two poultry mortality events in northwest Bangladesh, one in backyard poultry in Pabna in March 2017 and another in a commercial layer farm in Mymensingh in January 2018, to determine and molecularly characterize the etiological agent and to assess putative risk factors of disease outbreaks in both areas. We collected pooled swab samples from affected birds and environmental pooled swabs from affected and neighboring commercial poultry farms, backyard poultry, and live bird markets (LBM). We tested samples using specific real-time RT-PCR for influenza virus A (M gene), followed by HA and NA subtypes and sequenced and performed mutation analysis. We detected the H5N1 virus in the affected birds and all production and trading systems at both outbreak sites. Phylogenetic analysis of H5N1sequences revealed that the outbreaks were caused by reassortant of clade 2.3.2.1a H5N1 virus, which had also been detected in poultry at LBMs in Dhaka during an overlapping period. We identified several mutations likely to affect possible phenotypic changes, such as increased mammalian adaptation, reduced susceptibility to antiviral agents, and reduced host antiviral response. Investigation into poultry-keeping practices showed that most of the households, farms, and LBMs did not employ adequate biosecurity and hygiene, where (i) backyard poultry were typically reared in farmers’ dwellings, (ii) slaughter remnants were being discarded in the open where wild birds were scavenging waste, and (iii) farmers and vendors were generally not employing hand sanitizing after handling poultry. The investigation revealed that the relatedness of H5N1 strains across large spatial scales is high, suggesting a readily spreading and continuing evolution of the virus. Thus, the results contribute to sketching a bleak picture of the current AIV situation, the necessity for ongoing surveillance to track the evolution of H5N1 viruses in Bangladesh, and that adequate mitigation measures are urgently required.