Strontium isotopic ratios and Sr contents of molluscs from the Miocene Pebas Formation in Colombian Amazonia indicate that the bulk of the Pebas Formation was deposited in a freshwater environment, predominantly draining the Andes, with minor contribution from the shield regions. The molluscan oxygen and carbon isotopic composition does not provide a palaeosalinity proxy, but points to a lacustrine palaeoenvironment. The faunal character confirms such an interpretation. Sr isotopic ratios further suggest that a marine incursion that reached the area was composed of a mixture of shield-derived freshwater and seawater, with a maximum salinity of about 5 psu. Faunistic components of this incursion-level support a shield-seawater mixing. The incursion most likely reached western Amazonia from the north, probably along the western Guyana shield.