Background: Variations in the distribution and prevalence of pathogens in ticks can have significant consequences for human health. Information on these variables in Transcaucasia is scarce, so the aim of our study was to conduct a large-scale study to detect selected tick-borne infectious agents in Armenia. Methods: Overall, 209 adult ticks were collected from different hosts including 4 samples from human clothes. We tested ticks using high-throughput microfluidic single-cell real-time PCR to detect 42 genospecies of pathogens. We used GIS to determine biotic and abiotic factors governing the prevalence of pathogens and applied statistical analyses to test the association between prevalence of pathogens depending on hosts, locality and environment. Results: From 209 samples, 134 were positive to targeted pathogens. Anaplasma phagocytophilum Foggie, 1949 was the most prevalent case (44%). The highest overall prevalence was observed in ticks from sheep (74%), followed by cows (67%) and calves (60%). The highest multiple infection rates were also detected in sheep (40%) and calves (40%) followed by cows (28%). One statistically significant association was found among co-infections (P<0.05). The prevalence of pathogens varied according to locality. The abundance of Anaplasma spp. is significantly correlated with “slope” and “vegetation” factors. Similar patterns were detected for other pathogens. Conclusion: This was the first large-scale survey of multiple tick-borne pathogens in Armenia and Transcaucasia. The results of this study shed light on spatial variations in pathogen infection rate among adult ticks found on hosts and underline a number of environmental determinants of pathogen distribution among ticks.