How landscape composition and configuration impact the distribution of multi-vector and multi-host mosquito vector-borne disease systems, such as West Nile virus (WNV), remains challenging because of complex habitat and resource requirements by hosts and vectors that affect transmission opportunities. We examined correlations between landscape composition and configuration and 2018 WNV sentinel chicken seroconversion in Florida, USA across the state and within five National Oceanic Atmospheric Administration (NOAA) bioclimatic regions to understand strength and variation of landscape effects during an elevated transmission year. Although few landscape studies have examined WNV in Florida, we expected higher percentages of residential or medium-developed landscapes and more fragmented landscapes would be positively correlated with WNV seroconversion owing to the main mosquito vector habitats and avian host distributions. However, we expected to find variation in the importance of forest, wetland, and agriculture landscapes across bioclimatic regions in the state. WNV seroconversion rates were calculated using Florida 2018 Department of Health WNV sentinel chicken seroconversion data from 187 flocks maintained by mosquito control programs. Percent land cover and edge density metrics were calculated for multiple land cover classes and within multiple buffer distances from chicken coops using 2019 National Land Cover Data. We used binomial generalized linear mixed effects models to calculate the importance of landscape metrics to WNV seroconversion. We found no statewide predictors of seroconversion, but as expected, the importance of landscape varied across regions. In the north-central part of the state, we found higher seroconversion in less populated suburban areas while higher seroconversion in south-central Florida was correlated with fragmented forested areas within 0.5 km of coops and intact woody wetland areas within 2 km of coops. This work corroborates previous findings that consistent landscape predictors of WNV are difficult to identify across broader geographic areas and sets the stage for additional work that incorporates climate and landscapes interactions for a greater understanding of WNV ecology in this geographic region.