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IntroductionSpillover events ofMycoplasma ovipneumoniaehave devastating effects on the wild sheep populations. Multilocus sequence typing (MLST) is used to monitor spillover events and the spread ofM. ovipneumoniaebetween the sheep populations. Most studies involving the typing ofM. ovipneumoniaehave used Sanger sequencing. However, this technology is time-consuming, expensive, and is not well suited to efficient batch sample processing. MethodsOur study aimed to develop and validate an MLST workflow for typing ofM. ovipneumoniaeusing Nanopore Rapid Barcoding sequencing and multiplex polymerase chain reaction (PCR). We compare the workflow with Nanopore Native Barcoding library preparation and Illumina MiSeq amplicon protocols to determine the most accurate and cost-effective method for sequencing multiplex amplicons. A multiplex PCR was optimized for four housekeeping genes ofM. ovipneumoniaeusing archived DNA samples (N= 68) from nasal swabs. ResultsSequences recovered from Nanopore Rapid Barcoding correctly identified all MLST types with the shortest total workflow time and lowest cost per sample when compared with Nanopore Native Barcoding and Illumina MiSeq methods. DiscussionOur proposed workflow is a convenient and effective method for strain typing ofM. ovipneumoniaeand can be applied to other bacterial MLST schemes. The workflow is suitable for diagnostic settings, where reduced hands-on time, cost, and multiplexing capabilities are important.