A metagenomics approach was used to detect novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico. A total of 1359 mosquitoes of 7 species and 5 genera (Aedes, Anopheles, Culex, Mansonia and Psorophora) were sorted into 37 pools, homogenized and inoculated onto monolayers of Aedes albopictus (C6/36) cells. A second blind passage was performed and then total RNA was extracted and analysed by RNA-seq. Two novel viruses, designated Uxmal virus and Mayapan virus, were identified. Uxmal virus was isolated from three pools of Aedes (Ochlerotatus) taeniorhynchus and phylogenetic data indicate that it should be classified within the recently proposed taxon Negevirus. Mayapan virus was recovered from two pools of Psorophora ferox and is most closely related to unclassified Nodaviridae-like viruses. Two recognized viruses were also detected: Culex flavivirus (family Flaviviridae) and Houston virus (family Mesoniviridae), with one and two isolates being recovered, respectively. The in vitro host ranges of all four viruses were determined by assessing their replicative abilities in cell lines of avian, human, monkey, hamster, murine, lepidopteran and mosquito (Aedes, Anopheles and Culex) origin, revealing that all viruses possess vertebrate replication-incompetent phenotypes. In conclusion, we report the isolation of both novel and recognized RNA viruses from mosquitoes collected in Mexico, and add to the growing plethora of viruses discovered recently through the use of metagenomics.