Trout (Salmo trutta) exhibit anadromous and non-anadromous forms which are commonly sympatric. Offspring of the two forms can be separated by differences in characteristics such as stable isotope ratios and carotenoid pigments, which differ due to the influence of maternal resources. The rate of change in different characteristics due to freshwater feeding and the extent of differences between populations however remain unclear. Stable isotope (N and C) ratios and carotenoid pigment profiles were examined in the offspring of anadromous and non-anadromous fish sampled at different times from six sites within the catchment of the River Tweed, UK. Both techniques were able to separate newly emerged fry successfully, with carbon isotopes distinguishing the forms better than nitrogen isotopes and zeaxanthin being the primary carotenoid pigment used to distinguish the offspring of different migratory forms. By 4 months, stable isotope ratios of the two forms were still distinct although both carbon and nitrogen ratios needed to be considered to distinguish the forms. Zeaxanthin levels were more variable and overlapped between the offspring of the two forms. There was significant variation between populations and over time in both isotope ratios and carotenoid composition. Comparison between the two techniques would suggest that stable isotopes are more effective for distinguishing between offspring of different forms as the distinctions are evident for longer. Population differences in isotope ratios could influence the extent to which the forms can be distinguished and need to be quantified more thoroughly to fully evaluate the technique.