A conserved phase of gametogenesis is the development of oocytes and sperm within cell clusters (germline cysts) that arise through serial divisions of a founder cell. The resulting cell lineage trees (CLTs) exhibit diverse topologies across animals and can give rise to numerous emergent behaviors. Despite their centrality, sex-specific differences underlying the evolution and patterning of these cell trees are unknown. In Drosophila melanogaster, oocytes develop within a highly invariant and maximally branched 16-cell tree whose topology is constrained by the fusome – a branched membranous organelle critical for proper mitosis in females; the same division pattern and topology are widely thought to occur during spermatogenesis. Using highly-resolved three-dimensional reconstructions based on a supervised learning algorithm, we show that cell divisions in male cysts can deviate from the maximally branched pattern, leading to greater topological variability. Furthermore, in contrast to females, fusome fragmentation is common, suggesting germ cell mitoses can occur in its absence. These findings thus add to the repertoire of CLT formation strategies, highlighting the diversity of mechanisms employed during gametogenesis in the animal kingdom.SUMMARY STATEMENTDiegmiller et al. combine image processing techniques with analysis of cell network structure to identify sex-specific topological differences in germ cell lineage trees generation during Drosophila gametogenesis.