Background: Insecticide resistance is a growing threat to the efficacy of vector control interventions. Investigating the prevalence of molecular markers of resistance is a useful tool for monitoring the spread of resistance. The Bijagós Archipelago in Guinea-Bissau is a region of stable malaria transmission. However, the prevalence of molecular markers of resistance in malaria vectors is not well understood. Methods: High-throughput multiplex amplicon sequencing was used to investigate the prevalence of 17 different molecular markers associated with insecticide resistance in four genes: vgsc, rdl, ace1 and gste2. A total of 214 mosquitoes were analysed from 13 islands across the Bijagós. Results: Four of the 17 mutations screened for were identified in mosquitoes from the Bijagós: vgsc L995F (11.6%), N1570Y (6.1%) and A1746S (0.7%), and rdl A269G (1.1%). Bubaque is the most populous island in the archipelago, with the greatest population mobility and connection to continental Guinea-Bissau, and harboured higher frequencies of vgsc L995F and N1570Y mutations than the other islands. An additional 13 non-synonymous SNPs were identified across the four genes which have not been described previously. Conclusions: This study provides the first surveillance data for genetic markers present in malaria vectors from islands across the Bijagós Archipelago. Overall prevalence of insecticide resistance mutations is low. However, the identification of vgsc L995F and N1570Y mutations associated with pyrethroid resistance warrants further monitoring. This is particularly important as the mainstay of malaria control on the islands is the use of pyrethroid insecticide-treated nets.