Nestling feathers are often used to monitor and estimate Hg exposure in birds. Decision-making and results of studies on effects of exposure to Hg depend on adequate estimation of [Hg] at the studied level, which in turn is sensitive to variation in [Hg] in sampling units. However, there is little information on how feather [Hg] varies within nestlings, between nest mates, or between broods. We sampled feathers from sibling great egret nestlings in three breeding colonies of the Everglades where birds were exposed to variable Hg levels through diet. We compared the deviation of samples from average [Hg] within four levels of aggregation: individual (different scapular feathers of the same individual); brood (samples from siblings in the same nest), breeding colonies; and the entire wetland ecosystem. We found, on average, little intra-individual (textless2%) and intra-brood (textless6%) differences in [Hg], and no effects of chick hatch order in feather [Hg]. Further, intra-brood variability was not statistically different from intra-individual variability in [Hg]. In contrast, there was much higher variation between broods within colonies (textgreater20%) and within the entire wetland (textgreater30%) that could bias estimates of exposure to Hg obtained through small sample sizes. We simulated the influence of inter-brood variability in estimates of exposure to Hg under different sample sizes, and we found that uncertainty decreases markedly with samples sizes textgreater6, and suggest a sample size of 10 individuals from different nests for monitoring programs and to estimate Hg contamination in areas where no previous knowledge is available.