Guanabara Bay situated in Rio de Janeiro, Brazil, is a chronically contaminated tropical estuarine system surrounded by highly urbanized and industrialized area. Hydrocarbon inputs related to anthropogenic activities have been constant, including domestic and industrial wastes, petroleum industry activities, shipping traffic and deposition of vehicular emission, and fossil fuels combustion. Moreover, there was a major oil spill in January 2000, which released 1300m³ of marine heavy fuel oil in the Bay. Due to the multiple hydrocarbon sources and to transformations of original compounds under the tropical conditions, hydrocarbon source assignment is not trivial. The present chapter reports and evaluates three different approaches applied for source hydrocarbon identification in Guanabara Bay sediments collected between 2005 and 2007. One approach was based on gas chromatography - flame ionization detector (GC-FID) and gas chromatography - mass spectrometry (GC-MS) hydrocarbons quantitative analyses and the application of environmental geochemistry interpretation of the data, including diagnostic ratios and double log-log plots. The second approach applied multivariate analysis to the GC-MS quantitative data. The third approach was based on a combination of semiquantitative polycyclic aromatic hydrocarbon (PAH) analyses and application of multivariate data analysis of preprocessed sections of selected GC-MS chromatograms ("the CHEMSIC method"). All methodologies have shown their particular effectiveness for hydrocarbon source discrimination and source identification of PAHs in Guanabara Bay sediments.