For mammals with a polygynous mating system, dispersal is expected to be male-biased. However, with the increase in empirical studies, discrepancies are arising between the expected and observed direction/extent of the bias in dispersal. In this study, we assessed sex-biased dispersal in red deer (Cervus elaphus) on 13 estates from the Scottish Highlands. A total of 568 adult individuals were genotyped at 21 microsatellite markers and sequenced for 821 bp of the mitochondrial control region. Estimates of population structure with mitochondrial sequences were eight times larger than that obtained with microsatellite data (Fst′-mtDNA = 0.831; Fst′-micros = 0.096) indicating overall male-biased dispersal in the study area. Comparisons of microsatellite data between the sexes indicated a predominance of male-biased dispersal in the study area but values of FST and relatedness were only slighter larger for females. Individual-based spatial autocorrelation analysis generated a similar pattern of relatedness across geographical distances for both sexes, with differences only significant at two distance intervals (25–30 and 70–112 km). Patterns of relatedness differed between estates, male biased-dispersal was detected in eight estates but no sex-biased dispersal was found in the remaining five. Neither population density nor landscape cover was found to be associated with the patterns of relatedness found across the estates. Differences in management strategies that could influence age structure, sex ratio and dispersal behaviour are proposed as potential factors influencing the relatedness patterns observed. This study provides new insights on dispersal of a strongly polygynous mammal at geographical scales relevant for management and conservation.