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Theileria parasites are important tick-borne pathogens of animals and cause huge economic losses worldwide. Here, we undertook to assess the prevalence of Theileria spp. in cervids in Gansu, China, based on PCR analysis and sequencing of the 18S rRNA genes. Molecular survey showed that Theileria luwenshuni and Theileria sp. RSR were frequently found in Gansu cervids, and the prevalence of T. luwenshuni was 66.7 % ( n = 9, 6/9) in roe deer in Zhengning County, 58.3 % ( n = 12, 7/12) in sika deer in Ningxian County, 50 % ( n = 10, 5/10) in sika deer and 42.9 % ( n = 14, 6/14) in red deer in Weiyuan County. The prevalence of Theileria sp. RSR was 77.8 % ( n = 9, 7/9) in roe deer in Zhengning County, 75 % ( n = 12, 9/12) in sika deer in Ningxian County, 60.0 % ( n = 10, 6/10) in sika deer, and 50.0 % ( n = 14, 7/14) in red deer in Weiyuan County. The co-prevalence of the two T. luwenshuni and Theileria sp. RSR was 55.6 % ( n = 5, 5/9) in roe deer in Zhengning County, 50 % ( n = 12, 6/12) in sika deer in Ningxian county, 50 % ( n = 10, 5/10) in sika deer and 35.7 % ( n = 14, 5/14) in red deer in Weiyuan County, respectively. No other Theileria species was found in these samples. T. luwenshuni and Theileria sp. RSR were detected for the first time in cervids in Gansu, China. Animal experiments showed that four sheep were inoculated with roe deer blood from Zhengning, sika deer blood from Ningxian, sika deer blood, and red deer blood from Weiyuan, respectively, and the Theileria isolated from these inoculated sheep was identified as T. luwenshuni, but Theileria sp. RSR was not detected in these sheep. Our results extend our understanding of the epidemiology of cervine theileriosis in Gansu, China, and will facilitate the implementation of measures to control theileriosis in cervids and small ruminants. This was the first report to demonstrate the occurrence of T. luwenshuni from roe deer, sika deer, and red deer worldwide. [ABSTRACT FROM AUTHOR]