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Borreliosis is one of the most common vector-borne zoonotic diseases in the world. Limited data are available regarding Borrelia spp. and their genotypes in Kazakhstan. The goal of this study was to investigate the prevalence of Borrelia spp. in ixodid ticks collected in the southeastern region of Kazakhstan. A total of 1907 ixodid ticks were collected by flagging vegetation at three collection areas in the Almaty oblast between 2015 and 2018. They were grouped into 407 pools and examined by qPCR for Borrelia burgdorferi sensu lato (s.l.). A conventional PCR with specific primers targeting 16S rRNA gene was used to differentiate B. burgdorferi s.l. genospecies. Sequence analysis of the PCR products was performed for sixteen samples. Lyme borreliosis agents were only detected in adult questing Ixodes persulcatus. The overall B. burgdorferi s.l. prevalence in I. persulcatus estimated as the minimum infection rate reached 10.7 %. Borrelia burgdorferi sensu stricto was not detected in any of the tick pools. Partial 16S rRNA gene sequencing revealed the presence of B. miyamotoi, B. afzelii, and B. garinii. Borrelia afzelii was the dominant genospecies in Almaty oblast. A significantly lower proportion of B. garinii positive tick pools was detected in the Zailiyskiy Alatau as compared to the Dzungarian Alatau (χ 2 = 16.243; p = 0.0001) and Yenbekshikazakh district (χ 2 = 7.4156; p = 0.0065). The obtained results indicate the epidemiological significance of B. afzelii and B. garinii in southeastern Kazakhstan. These new data aim to improve the diagnostics of Lyme borreliosis and monitoring of tick-borne infections in Kazakhstan., (Copyright © 2021. Published by Elsevier GmbH.)