Microplastic (MP) contamination in freshwater systems has emerged as a growing environmental concern. This study investigated the occurrence and seasonal variability of MPs in surface water, the water column, and sediments at selected sites in Lake Alhajuela, Panama. Lake Alhajuela is an artificial reservoir that supplies water to the Panama Canal lock system and to the cities of Panama and Colón, serving more than 50% of the country’s population. MPs were isolated using two digestion protocols followed by density separation, and fragments and films larger than 1 mm were chemically characterized using FTIR–ATR spectroscopy. Mean MP concentrations were 759 ± 536 MPs L⁻¹ in surface water, 328 ± 140 MPs L⁻¹ in the water column, and 109 ± 87 MPs g⁻¹ in sediments. Statistical analyses revealed no significant differences among sampling sites; however, significant seasonal differences were observed (p < 0.01). Smaller MPs (63–249 µm) were more abundant compared to larger MPs (>250 µm). Fragments and fibers were the most predominant type of MP reported. Our results confirm the presence of MPs in the surface and water column, as well as sediments of the Alhajuela Lake. Further studies are needed to elucidate the fate, sources, transport, and distribution of MPs across Lago Alhajuela as well as to assess the lake’s potential contribution of MPs to Gatun Lake and the Panama Canal system.