<title>Abstract</title> This study aimed to describe the diversity of ixodid tick species in rural forest areas of Yopal and Aguazul, Department of Casanare, Colombia, and to evaluate the presence of tick-borne <italic>Rickettsia</italic> sp., potentially impacting animal and human health. A cross-sectional study was conducted on questing ticks collected from forested farm areas that keep livestock and/or conduct agricultural activities. All ticks were subjected to DNA extraction and tested by PCR amplifying fragments of three rickettsial genes: <italic>gltA</italic>, <italic>htrA</italic>, and <italic>ompA</italic>. A total of 852 individual ticks (larvae, nymphs, and adults) and 15 additional larvae clusters were collected and identified as <italic>Amblyomma mixtum</italic>, <italic>Amblyomma</italic> cf. <italic>parvum</italic>, <italic>Amblyomma dissimile</italic>, <italic>Amblyomma</italic> spp., and <italic>Dermacentor</italic> spp. <italic>Rickettsia</italic> organisms were detected by PCR in 7.0% (16/230) of the individual larvae, 0.2% (1/488) of the nymphs, and 1.5% (2/134) of the adults. “<italic>Candidatus</italic> Rickettsia colombianensi” and <italic>Rickettsia amblyommatis</italic> were detected in 1.52% (13/852) and 0.47% (4/852) of the ticks, respectively. In addition, a novel genotype, <italic>Rickettsia</italic> sp. genotype Yopal, phylogenetically closest to <italic>Rickettsia canadensis</italic>, was identified in two <italic>A. mixtum</italic> males. Several tick and <italic>Rickettsia</italic> species were found in the study areas, suggesting the need for an in-depth study of rickettsiosis in the Department of Casanare and across Colombia. Priority areas for future studies include investigating the molecular, clinical, and epidemiological features of the presentation of tick species and the possible participation of “<italic>Ca</italic>. R. colombianensi”, <italic>R. amblyommatis</italic>, and “<italic>Rickettsia</italic> sp. Yopal genotype of <italic>A. mixtum</italic>” to understand their role in human rickettsiosis in the study region.