Germline mutation rate (GMR) is a key concept in all disciplines of biology, particularly in the biomedical sciences and evolutionary biology. While GMR varies within species, the factors driving this variation remain unclear. Two primary mechanisms—replication-driven and damage-induced mutations—are thought to contribute to this variation. This study aims to quantify the relative contributions of these mechanisms using the leafcutter ant Acromyrmex echinatior, a model well-suited for this investigation due to its unique reproductive biology: 1. Maternal replication-driven mutations: Queen ants are long-lived (~20 years) and highly fertile, making maternal germline mutations likely to be replication-driven. 2. Paternal damage-induced mutations: Queens mate with 4–5 males, store sperm for life, and never remate. Males, however, die within days of mating, meaning paternal germline mutations must arise before fertilization, likely due to DNA damage. 3. Controlled laboratory system: Routine sampling of mother-offspring pedigrees allows precise control of parental age and enables haplotype-based resolution of maternal versus paternal mutation rates. This study will enable the direct estimation of the relative contributions of different mutational mechanisms to GMR variation. By shedding light on the factors influencing mutation rates, it will provide novel insights into their evolutionary consequences.