Pimelodus grosskopfii is an important catfish in inland fisheries in Colombia as well as an attractive species for aquaculture. The aim of this study was to clone and determine the coding sequence of the GP-α and LHβ subunits of the luteinizing gonadotropin of this species. Total RNA from pituitary glands was extracted and cDNA was synthesized. GP-α and LHβ purified PCR products were linked to pJET1.2/blunt® followed by Escherichia coli DH5α® transformation. The extracted plasmids of the positive clones were sequenced. The open reading frame of GP-α and LHβ subunits was 351 bp (116 amino acids) and 423 bp (140 amino acids) respectively. The GP-α subunit showed high degrees of homology (amino acids) with GP-α subunit from different families of Siluriformes (87-99%) and lower homology with teleost orders such as Perciformes, Salmoniformes and Cypriniformes (66-90%). Similarly, the homology (amino acids) of LHβ subunit was high within siluriformes (91-97%) and lower with other orders (65-86%). The results obtained in the present study, provide basic information on the sequence of gonadotropins in neotropical catfishes and can be used for the development of recombinant hormones to optimize reproductive control in P. grosskopfii.