Stable isotope mixing models are regularly used to provide probabilistic estimates of source contributions to dietary mixtures. Whilst Bayesian implementations of isotope mixing models have become prominent, the use of appropriate diet-tissue discrimination factors (DTDFs) remains as the least resolved aspect. The DTDFs are critical in providing accurate inferences from these models. Using both simulated and laboratory-based experimental data, this study provides conceptual and practical applications of isotope mixing models by exploring the role of DTDFs. The experimental study used Mozambique Tilapia Oreochromis mossambicus, a freshwater fish, to explore multi-tissue variations in isotopic incorporation patterns, and to evaluate isotope mixing model outputs based on the experiment- and literature-based DTDFs. Isotope incorporation patterns were variable for both muscle and on tissues among the consumer groups that fed diet sources with different stable isotope values. Application of literature-based DTDFs in isotope mixing models consistently underestimated the dietary proportions of all single-source consumer groups. In contrast, application of diet-specific DTDFs provided better dietary estimates for single-source consumer groups. Variations in the proportional contributions of the individual sources were, nevertheless, observed for the mixed-source consumer group, which suggests that isotope assimilation of the individual food sources may have been influenced by other underlying physiological processes. This study provides evidence that stable isotope values from different diet sources exhibit large variations as they become incorporated into consumer tissues. This suggests that the application of isotope mixing models requires consideration of several aspects such as diet type and the associated biological processes that may influence DTDFs.