The taxonomic study of the genus Opuntia was carried out in the municipalities of Los Santos and Piedecuesta (Police Inspection of Pescadero), Santander–Colombia. Opuntia is represented by nine species distributed in sub xerophytic scrubs and dry forests, between 576 to 1867 m a.s.l. A key is proposed for the identification of the species, a taxonomic sheet was elaborated with the following information: iconographies, extended descriptions, distribution, habitat and revisions of herbarium material. In addition, five neotipifications are proposed: Opuntia caracassana Salm–Dyck, O. pubescens H. L. Wendl. ex Pfeiff, O. quitensis F.A.C. Weber, O. schumannii F.A.C.Weber ex A.Berger and O. wentiana Britton & Rose (= Opuntia caracassana ). Opuntia pittieri and O. pennellii are endemic to Colombia.