Adequate genetic variation in a population is fundamental to reducing the risk of inbreeding depression in the short term and maintaining its ability to respond to evolutionary forces over the long term. There are now several studies that use microsatellite loci to assess genetic variation within populations across the geographic range of Emydoidea blandingii (Blanding's Turtle), but direct comparisons among studies have not been conducted. We present estimates of allelic richness (A) and expected heterozygosity (H_E) from 59 localities across much of the species'geographic range and compare them using a resampling method that calibrates values relative to a reference population to account for different sample sizes and microsatellite panels. We further compared these measures between 2 datasets, from the midwestern US and from the northeastern US, that were made compatible using a sample of re-genotyped individuals. In both cases, we found lower A and H_E in the northeastern localities. We also developed a sensitivity analysis of effective population size (N_e) estimation with the linkage disequilibrium method (N_eLD) that used a well-sampled population modelled in the program ‘NeOGen’ to explore the effects of adult population size (N_c), sample size, and locus number while accounting for overlapping generations, life history, and demography. We find that it is possible to estimate N_e with accuracy and precision in populations with N_c < 400 when sample size is ∼25% of adult population size and ≥11 microsatellite loci are used. With this benchmark, we then estimated N_eLD in 7 localities using single-sample linkage disequilibrium in NeEstimator, a method that does not account for overlapping generations, demography, and life history but is more accessible for users. This approach overestimated N_eLD by ∼37%. Collectively, our analyses of genetic variation within populations facilitate assessment of population status and resiliency in Blanding's Turtles by demonstrating the range of genetic variation across a large number of populations and developing a baseline for the estimation of effective population size in natural populations.