Antimicrobial resistance is recognized worldwide as one of the greatest threats to human and animal health and the environment. To evaluate the resistance rate of Gallibacterium anatis biovar haemolytica, which contributes to acteremia, oophoritis, ovarian follicle degeneration, salpingitis,decreased egg production, and increased mortality in hens, strains isolated from the reproductive tracts of layers were analyzed. The oviducts were taken from three hens from each of 10 flocks manifesting clinical signs related to laying. Twenty-two isolates of G. anatis biovar haemolytica collected from the three parts of the reproductive system were identified using MALDI-TOF and molecular methods. The biovar’s resistance to 19 antimicrobial substances was assessed using the disk diffusion (n = 8) and broth microdilution (n = 11) methods. The presence of virulence (gtxA, gyrB, and flfA) and antibiotic resistance (blaROB, aphA, tetB, and tetH) genes was examined using PCR. All the isolates were resistant to four or more classes of antibiotics and were considered multidrug-resistant. All such isolates were resistant to tilmicosin, tylosin, and enrofloxacin, 88.2% were to tetracycline, and 82.4% to vancomycin. The gtxA, gyrB, tetB, and tetH genes were demonstrated. Considering the present prevalence of multidrug resistance among G. anatis biovar haemolytica isolates from laying hen reproductive tracts, surveillance in reproductive flocks is warranted.