Introduction: Chick embryo has been historically and globally a research model in human medicine including regenerative medicine, cancer, drug delivery testing. Compared to other animal models, chick embryo is unexpensive, easy manipulation and has rapid development of the embryo. In Vietnam, implementing chick embryo in vaccines production and pharmaceuticals has been utilized. Therefore, maintaining sufficient sources of eggs that can be used in animal model research is important. During egg incubation, several factors could affect the mortality of the embryo, such as the environment, the nutrition, and the pathogenic infection. However, recent data illustrated that 25 kb genomic deletion containing Indian Hedgehog gene and Non-Homologous End-Joining 1 gene was also the cause for chick embryo lethality. IHH functions in bone formation, skeletal morphogenesis, while NHEJ1 is vital for repairing DNA double strand breakage. Individuals carrying two copies of 25 kb deletion are lethal at birth. As a result, genetic testing is needed to ensure embryonic survival. Methodology: In this paper, we aimed to develop a multiplex PCR test to detect the 25 kb deletion in three indigenous Vietnamese chicken breeds: Phu Phan, Hmong, and Ri. To our best knowledge, this is the first study in Vietnam to address the avian embryo mortality in Vietnamese chicken populations. Results: Three primers PCR assay was successfully established and optimized. Our data showed that the frequency of lethal allele in Phu Phan, Hmong and Ri was 2%, 3% and 5%, respectively. Conclusion: In short, our newly established assay provides an efficient, trustworthy, and practical tool for 25 kb deletion carrier detection but also promotes a monitoring system for chicken embryos.