Objective To understand the epidemiological characteristics of antimicrobial resistance genes in clinically isolated Enterobacterales in a region, and to analyze the model of clonal transmission and horizontal transfer of antimicrobial resistance genes in resistant strains. Methods 133 clinical isolates of Enterobacterales from blood and fecal specimens from a sentinal hospital in Jiamusi from March 2023 to October 2024 were collected for antimicrobial susceptibility testing and whole genome sequencing. Analyses of resistance gene and plasmid replicon, multilocus sequence typing (MLST), and phylogenetic analysis were conducted based on genomic data. Horizontal transfer ability of resistant plasmids was evaluated through conjugation transfer test. Results In Klebsiella pneumoniae, bla_SHV (93.68%), oqxAB (97.89%), and fosA (86.32%) were the most frequently detected resistance genes, while ST23 was the dominant epidemic type (30.53%), with a percentage of 61.7% in the IncH (pNDM-MAR) plasmid. In diarrheal Escherichia coli DEC, bla_CTX-M (57.69%), bla_TEM (61.54%), and qnr (38.46%) were the main resistance genes. IncF plasmids were widely present (92.31%), and 80.00% of donor strains exhibited the ability to conjugate and transfer resistant plasmids. Conclusion The transmission of Enterobacterales in this region may be driven by two modes: ST23 IncH (pNDM-MAR) complex clone expansion and IncF plasmid horizontal transfer. Strengthening molecular monitoring of high-risk clone-plasmid complex is crucial for controlling the transmission of healthcare-associated infection.