Most of sequenced West Nile virus (WNV) genomes encode a single N-linked glycosylation site on their envelope (E) proteins. We previously found that WNV lacking the E protein glycan was severely inhibited in its ability to replicate and spread within two important mosquito vector species, Culex pipiens and Cx. tarsalis. However, recent work with a closely related species, Cx. pipiens pallens, found no association between E protein glycosylation and either replication or dissemination. To examine this finding further, we expanded upon our previous studies to include an additional Culex species, Cx. quinquefasciatus. The non-glycosylated WNV-N154I virus replicated less efficiently in mosquito tissues after intrathoracic inoculation, but there was little difference in replication efficiency in the midgut after peroral infection. Interestingly, although infectivity was inhibited when WNV lacked the E protein glycan, there was little difference in viral spread throughout the mosquito. These data indicate that E protein glycosylation affects WNV-vector interactions in a species-specific manner.