Spatial and temporal patterns of frequency variation for a neutralized B chromosome in the grasshopper Eyprepocnemis plorans were analyzed along six transects in the east of Spain to explore possible factors affecting the population dynamics of this polymorphism. Three parameters were employed to quantify B frequency: prevalence, load and mean frequency. Of them, load seemed to be the less sensitive parameter, probably due to its small range of variation. Prevalence, however, shows ample variation, but the mean frequency of B chromosomes per individual is the best parameter to characterize B frequency. Only river transects revealed significant differences among populations, and the use of two geographic explicit approaches (Mantel test and distograms) revealed significant isolation by distance (IBD), especially at the Segura River mouth, presumably due to low gene flow and drift. No temporal trend was found in the Segura River transects, which is consistent with the slow changes in B frequency expected during the random walk for neutralized B chromosomes. But these transects showed a clear spatial pattern, with B1 showing lower frequency in the upper course of this river. The present results provide the first empirical evidence of IBD in the evolution of a neutralized B chromosome, and support the notion that B dynamics at this evolutionary stage is best explained by a metapopulation approach.