AbstractIn citrus, development of new hybrids is difficult due to failure of the germination of zygotic embryos. Hence, this study was conducted to standardize embryo age and media for maximizing the germination and subsequent seedling growth in sour orange × Sacaton citrumelo crosses followed by the identification of simple sequence repeats (SSR) markers for distinguishing the hybrids. A factorial experiment in complete randomized design was conducted with two each embryo age (110–120 and 130–140 days after pollination) and culture media (G‐B5 supplemented with 1.5 mg/L GA3 plus malt extract and G‐B5 plus malt extract) for in vitro embryo culture of sour orange × Sacaton citrumelo progenies. Older embryos germinated well in both media, though highest on M1 medium (100%). In fact, M1 medium gave higher plantlet survival in both age groups. The seedling growth of older embryos had highest root length (37.80 mm) and shoot length (24.20 mm) in M1 medium at 60 days after inoculation (DAI). Three SSR markers (TAA45, CAC15 and CAC39) showed polymorphism between female and male parents and were able to identify their hybrids.