We review the recently developed local spatial autocorrelation statistics Ii, ci, Gi, and Gi*. We discuss two alternative randomization assumptions, total and conditional, and then newly derive expectations and variances under conditional randomization for Ii and ci, as well as under total randomization for ci. The four statistics are tested by a biological simulation model from population genetics in which a population lives on a 21 × 21 lattice of stepping stones (sixty-four individuals per stone) and reproduces and disperses over a number of generations. Some designs model global spatial autocorrelation, others spatially random surfaces. We find that spatially random designs give reliable test results by permutational methods of testing significance. Globally autocorrelated designs do not fit expectations by any of the three tests we employed. Asymptotic methods of testing significance failed consistently, regardless of design. Because most biological data sets are autocorrelated, significance testing for local spatial autocorrelation is problematic. However, the statistics are informative when employed in an exploratory manner. We found that hotspots (positive local autocorrelation) and coldspots (negative local autocorrelation) are successfully distinguished in spatially autocorrelated, biologically plausible data sets.