In recent years, the problem of microplastic pollution has begun to receive more attention. Currently, it is known that these particles, less than 5 mm in diameter, can lead to problems for both ecosystems and human health due to the toxicity of their components. In spite of this, research on this topic has focused mainly on the oceans, leaving aside rivers, which are the main source of these pollutants to oceans. Additionally, information is limited to certain rivers in countries of the northern hemisphere where wastewater treatment plants can retain up to 80% of microplastics. In South America, microplastic pollution is practically unknown, and wastewater treatment in several areas is still limited. This study focused on quantifying the microplastics present in the upper basin of the Guayllabamba River, in the Tropical Andes, a biodiversity hotspot. This basin is where the capital city of Ecuador, Quito, is located. Less than 10% of the wastewater in Quito is treated and the rest is dumped to rivers without treatment. We performed a physical analysis of microplastics, by weight and by category of microplastic, in various sampling points before and after urban areas. We found microplastic pollution beginning in the headwaters of the basin, with significant increases in urban areas of the Metropolitan District of Quito. Values of suspended microplastics in rivers after urban areas were higher than those recorded in the literature. Plastic levels in sediment were also higher after urban areas. Microplastics were highly correlated with other water pollutants, showing the prevailing necessity of wastewater treatment plants, because all of this pollution is dumped into rivers that flow from 2800 m a.s.l. to highly diverse freshwater ecosystems and human populations located downstream that depend on these aquatic sources, and finally to the Pacific Ocean.