Stable isotopes offer an accurate and cost-effective way to understand critical pathways of energy, and their use has application where aquatic habitats have been degraded. Isotope ratios can offer insights into appropriate conservation and rehabilitation techniques to manage these valuable resources. To date, the literature shows little attention to spatial and temporal variation in isotope signatures of samples taken from estuarine environments. This study reports on an investigation of sample size by which to determine isotopic signatures within a single species of fish, Ambassis jacksoniensis. Provided are two ways by which to determine how many samples are needed, and the results imply that care may be needed in interpreting previous studies based on small sample size. A minimum of five individuals would appear necessary in some cases.