Abstract.1The yellow subterranean antLasius flavusis generally considered to have a single queen in mature nests. Yet, many anecdotal observations have suggested that the conspicuous earth mounds of this ant may, at least occasionally, contain several queens and their offspring.2To estimate the number of matrilines per mound, samples for horizontal starch gel electrophoresis were collected from a total of thirty‐seven mounds in three sites of old chalk‐grassland in southern England.3Allozymatic variation at an esterase and hexokinase locus showed that probably more than 50% of the mounds contained more than one queen and that the mean number of queens per mound is at least 1.5–2.4Further results strongly suggested that workers from different matrilines may share the same galleries, at least in the top of the mound.5The apparently variable social structure ofLasius flavuscolonies is discussed, both technically and with reference to ecological processes related to the presence of ants in the nest m