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American foulbrood (AFB) is an infectious disease of honey bee brood caused by the endospore-forming bacterium Paenibacillus larvae. P. larvaespores are resilient in the environment, thus colonies with clinical signs of AFB are often destroyed by burning to eradicate the causative agent. To prevent outbreaks of AFB, oxytetracycline metaphylaxis is widely used in North America, resulting in sustained selective pressure for oxytetracycline resistance in P. larvae. To determine if antimicrobial resistance (AMR) is present among P. larvaeisolates from commercial beekeeping operations in Saskatchewan, Canada, we performed antimicrobial susceptibility testing of 718 P. larvaesamples cultured from pooled, extracted honey collected from 52 beekeepers over a 2-y period, 2019 and 2020. We found that 65 of 718 (9%) P. larvaesamples collected from 8 beekeepers were resistant to oxytetracycline with minimum inhibitory concentration (MIC) values of 64–256 µg/mL. Eight of 718 (1%) samples from 4 beekeepers had intermediate resistance to oxytetracycline (MIC: 4–8 µg/mL). Susceptibility testing for tylosin and lincomycin indicated that P. larvaein Saskatchewan continue to be susceptible to these antimicrobials (tylosin MIC: <1 µg/mL, lincomycin MIC: ≤2 µg/mL). Most oxytetracycline-resistant P. larvaesamples were identified in northeastern Saskatchewan. Whole-genome sequence analysis identified the P. larvae–specific plasmid pMA67with tetracycline-resistance gene tet(L) in 9 of 11 oxytetracycline-resistant P. larvaeisolates sequenced. Our results highlight the advantage of using pooled, extracted honey as a surveillance tool for monitoring AMR in P. larvae.