Bio-optical variables in the Northern Gulf of California were analyzed using in situ and satellite data obtained during a cruise in June 2008. The study area was divided into three bio-optical regions: Upper Gulf (UG), Northern Gulf (NG), and Great Isles (GI). Each region was characterized according to phytoplankton pigment concentration, phytoplankton and nonpigmented material spectral absorption coefficients, and spectral reflectance. Observed patterns were an indication of the shift in bio-optical conditions from north to south going from turbid and eutrophic waters to mesotrophic ones. Although there was a good agreement between satellite and in situ Chl a (RMSE ±33%), an overestimation of in situ Chl a was observed. This was partly explained by the presence of nonalgal particles, as well as the influence of desert and continental aerosols, which is generally overcorrected in the standard processing. The UG and NG could be considered as Case 2 waters, but they did exhibit different bio-optical characteristics. This implies that both biological and optical properties should be invoked to better understand water reflectance variability in the study region and its implications for the remote sensing of Chl a and biogeochemical processes.