BACKGROUND Though most of Panamá is free from malaria, localized foci of transmission persist, including in the Guna Yala region. Government-led entomological surveillance using an entomological surveillance planning tool (ESPT) sought to answer programmatically-relevant questions that would enhance the understanding of both local entomological drivers of transmission and gaps in protection that result in persisting malaria transmission to guide local vector control decision-making. METHODS The ESPT was used to design a sampling plan centered around the collection of minimum essential indicators to investigate the relevance of LLINs and IRS in the communities of Permé and Puerto Obaldía, Guna Yala, as well as to pinpoint any remaining spaces and times where humans are exposed to Anopheles bites (gaps in protection). Adult Anopheles were collected at three time points via human landing catches (HLCs), CDC Light Traps (LT), and pyrethrum spray catches (PSCs) during the rainy and dry seasons. Mosquitoes were identified to species via molecular methods. Insecticide susceptibility testing of the main vector species to fenitrothion was conducted. RESULTS In total, 7537 adult Anopheles were collected from both sites. Of the 493 specimens molecularly confirmed to species, two thirds (n = 340) were identified as Nyssorhynchus albimanus, followed by Anopheles aquasalis. Overall Anopheles human biting rates (HBRs) were higher outdoors than indoors, and were higher in Permé than in Puerto Obaldía: nightly outdoor HBR ranged from 2.71 bites per person per night (bpn) (Puerto Obaldía), to 221.00 bpn (Permé), whereas indoor nightly HBR ranged from 0.70 bpn (Puerto Obaldía) to 81.90 bpn (Permé). Generally, peak biting occurred during the early evening. The CDC LT trap yields were significantly lower than that of HLCs and this collection method was dropped after the first collection. Pyrethrum spray catches resulted in only three indoor resting Anopheles collected. Insecticide resistance (IR) of Ny. albimanus to fenitrothion was confirmed, with only 65.5% mortality at the diagnostic time. CONCLUSIONS The early evening exophagic behaviour of Anopheles vectors, the absence of indoor resting behaviours, and the presence of resistance to the primary intervention insecticide demonstrate limitations of the current malaria strategy, including indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs), and point to both gaps in protection and to the drivers of persisting malaria transmission in Guna Yala. These findings highlight the need for continued and directed entomological surveillance, based on programmatic questions, that generates entomological evidence to inform an adaptive malaria elimination strategy.