Microplastics (MPs) are a ubiquitous pollutant, emphasizing the need to understand their abundance and the factors that influence these patterns around the globe. In a prior study, high numbers of MPs were found in surface waters and tissues of the oyster Crassostrea virginica collected from one location in the Indian River Lagoon (IRL, FL, USA). To better understand spatial and temporal variability of MPs throughout the IRL, for one year, monthly surface water samples were collected from 35 sites, while oysters were collected quarterly from 12 sites. Microscopy and ATR-FTIR were used to quantify MP. In total, 3755 MPs were found in 44% of water samples (mean density ± CI: 1.47 ± 0.09 MP/L). South IRL water had the most MPs, likely associated with proximity to urbanization, inlets (MP sinks) and tributaries (MP sources). MP (n = 3181) were found in 70% of examined C. virginica (n = 1402). Abundances of MP in oysters were lower in the spring and in north IRL. The overall mean abundance was 2.26 ± 0.16 MP/oyster, and the density was 2.43 ± 0.52 MP/g wet tissue weight. Our results provide a more complete picture of MPs in the IRL, a subtropical, shallow-water estuarine system.