Some walnut trees (Juglans regia L.) originating from central Asia display an early flowering phenotype. These “Early Mature” (EM) trees may produce flowers within months of germination. Secondary flowering waves are also observed within a growing season. Inflorescences may carry male, female and hermaphrodite flowers. Progeny obtained from selected EM trees were cultured in vitro to initiate clonal propagation of these genotypes. Embryogenic lines were established through the culture of immature zygotic embryos. Microshoot lines were obtained from germinated somatic or zygotic embryos. Plants showing EM phenotypes were recovered through direct conversion of somatic embryos or adventitious rooting of microcuttings. During the in vitro propagation phase, flower buds were observed on microshoots after three to six subcultures. Histological analysis showed that most of these flowers were hermaphrodite. In vitro apical buds were used to clone the walnut orthologous cDNAs of the AGAMOUS and APETALA 3 MADS-box genes. Northern blots revealed a preferential expression of both of these homeotic genes in flowers. The results highlight the usefulness of EM lines to study the genetic cues controlling flowering and sexual maturity in woody perennials.