We report strontium isotopic results for the late Miocene Hualapai Limestone of the Lake Mead area (Arizona-Nevada) and the latest Miocene to early Pliocene Bouse Formation and related units of the lower Colorado River trough (Arizona-California-Nevada), together with parallel oxygen and carbon isotopic analyses of Bouse samples, to constrain the lake-overflow model for integration of the Colorado River. Sr isotopic analyses on the basal 1–5 cm of marl, in particular along a transect over a range of altitude in the lowest-altitude basin that contains freshwater, brackish, and marine fossils, document the 87Sr/86Sr of first-arriving Bouse waters. Results reinforce the similarity between the 87Sr/86Sr of Bouse Formation carbonates and present-day Colorado River water, and the systematic distinction of these values from Neogene marine Sr. Basal Bouse samples show that 87Sr/86Sr decreased from 0.7111 to values in the range 0.7107–0.7109 during early basin filling. 87Sr/86Sr values from a recently identified marl in the Las Vegas area are within the range of Bouse Sr ratios. 87Sr/86Sr values from the Hualapai Limestone decrease upsection from 0.7195 to 0.7137, in the approach to a time soon after 6 Ma when Hualapai deposition ceased and the Colorado River became established through the Lake Mead area. Bouse Formation δ18O values range from –12.9‰ to +1.0‰ Vienna Pee Dee belemnite (VPDB), and δ13C between –6.5‰ and +3.4‰ VPDB. Negative δ18O values appear to require a continental origin for waters, and the trend to higher δ18O suggests evaporation in lake waters. Sr and stable isotopic results for sectioned barnacle shells and from bedding planes of the marine fish fossil Colpichthys regis demonstrate that these animals lived in saline freshwater, and that there is no evidence for incursions of marine water, either long-lived or brief in duration. Lack of correlation of Sr and O isotopic variations in the same samples also argue strongly against systematic replacement of Sr in Bouse carbonates after deposition. Our results reinforce the conclusion that the Bouse Formation was deposited in a descending series of basins connected by overflow of Colorado River water. The Hualapai Limestone records a separate and earlier lake that may have been progressively influenced by Colorado River water as the time of river integration approached.