Anopheles mosquitos are the primary vectors for the rapid spread of malaria in Limpopo, South Africa, negatively impacting all malaria elimination agenda; particularly for a country with vector control as one of the major malaria elimination strategies. The implementation of vector control strategies, however, depends on the accurate identification of mosquito vectors, which has been lacking in Limpopo as a result of earlier studies that mainly relied on morphological identification of vectors, which has numerous drawbacks that lead to misidentification of mosquitoes, thus resulting in the development and assignment of ineffective control strategies. All this together set back elimination strategies and programs. The present study therefore aimed to molecularly identify mosquitoes collected from various regions within Limpopo province. This was accomplished by examining genetic composition of mosquitos from different regions within Limpopo province, using molecular genetics techniques such as 18S rDNA-based PCR analysis, 18S rDNA sequencing, etc. This investigation was successful since 18S rDNA was able to identify every one of the 42 mosquito samples that were analysed, revealing that the samples belonged to 7 different Anopheles species and the majority of the species were molecularly demonstrated to have been misidentified morphologically. The outcomes of the molecular analysis, which involved comparing the query sequences to the sequences in GenBank, were supported by the close relationship among mosquitoes of the same species in maximum likelihood trees and the presence of high to 100% bootstrap support values, giving confidence to conclude that mosquitoes of the same species under study are genetically related. Due to a number of constraints covered in this study, morphological identification of mosquitoes is prone to misidentification of species, hence genetic characterization is recommended to be used as a confirmatory approach in the identification of the species.