AbstractVairimorpha ceranae and Vairimorpha apis are widespread microsporidian pathogens of honey bees, yet information on their occurrence in the indigenous Arabian honey bee, Apis mellifera jemenitica, remains limited. This study investigated the prevalence and seasonal dynamics of Vairimorpha spp. in A. m. jemenitica colonies from arid and semi-arid regions of Saudi Arabia. Nine regions were screened using species-specific PCR, while monthly microscopic spore counts assessed seasonal dynamics over one year. V. ceranae showed significantly higher prevalence (77.8% to 100%) than V. apis (11%) within both climatic zones (Fisher’s exact test, P < 0.001). Pairwise comparisons revealed significant localized variation among most regions in V. apis prevalence (Fisher’s exact test, P > 0.05). Co-occurrence analysis demonstrated a significant positive association between V. ceranae and V. apis (ϕ = 0.42, P = 0.003), with all V. apis infections occurring as co-infections with V. ceranae. Seasonal spore loads differed significantly among months (ANOVA, F = 2.47, p = 0.015), declining during spring and reaching a maximum in December, while remaining relatively low overall (3.0 × 104–3.2 × 105 spores per bee). These findings demonstrate the widespread establishment of V. ceranae in A. m. jemenitica populations, the restricted distribution of V. apis, and relatively low infection intensity (spore count/bee) under Saudi Arabian conditions, providing a baseline for future investigations of host–pathogen interactions and disease ecology in A. m. jemenitica under challenging environmental conditions.