For organisms with overlapping generations that occur in metapopulations, uncertainty remains regarding the spatio-temporal scale of inference of estimates of the effective number of breeders (N^b)and whether these estimates can be used to predict generational Ne. We conducted a series of tests of the spatio-temporal scale of inference of estimates of Nb in nine consecutive cohorts within a long-term study of brook trout (Salvelinus fontinalis). We also tested a recently developed approach to estimate generational Ne from N^b and compared this to an alternative approach for estimating N^e that also accounts for age structure. Multiple lines of evidence were consistent with N^b corresponding to the local (subpopulation) spatial scale and the cohort-specific temporal scale. We found that at least four consecutive cohort-specific estimates of N^b were necessary to obtain reliable estimates of harmonic mean N^b for a subpopulation. Generational N^e derived from cohort-specific N^b were within 7 - 50% of an alternative approach to obtain, N^e suggesting some population-specificity for concordance between approaches. Our results regarding the spatio-temporal scale of inference for Nb should apply broadly to many taxa that exhibit overlapping generations and metapopulation structure and point to promising avenues for using cohort-specific N^b for local-scale genetic monitoring.