Anthropogenic fecal pollution in urban waterbodies can promote the spread of waterborne disease. The objective of this study was to test crAssphage, a novel viral human fecal marker not previously applied for fecal source tracking in Latin America, as a fecal pollution marker in an urban river in Chile. Human fecal markers crAssphage CPQ_064 and Bacteroides HF183, the human pathogen norovirus GII, and culturable fecal indicator bacteria (FIB) were quantified at six locations spanning reaches of the Mapocho River from upstream to downstream of Santiago, as well as in repeated sub-daily frequency samples at two urban locations. Norovirus showed positive correlation trends with crAssphage (τ = 0.57, p = 0.06) and HF183 (τ = 0.64, p = 0.03) in river water, but not with E. coli or enterococci. CrAssphage and HF183 concentrations were strongly linearly related (slope = 0.97, p < 0.001). Chlorinated wastewater effluent was an important source of norovirus GII genes to the Mapocho. Precipitation showed non-significant positive relationships with human and general fecal indicators. Concentrations of crAssphage and HF183 in untreated sewage were 8.35 and 8.07 log10 copy/100 ml, respectively. Preliminary specificity testing did not detect crAssphage or HF183 in bird or dog feces, which are predominant non-human fecal sources in the urban Mapocho watershed. This study is the first to test crAssphage for microbial source tracking in Latin America, provides insight into fecal pollution dynamics in a highly engineered natural system, and indicates river reaches where exposure to human fecal pollution may pose a public health risk.