Primitive hematopoiesis is a crucial process in the organism, responsible for the transportation of oxygen and nutrients during early embryonic stages and laying the foundation for the immune system. During primitive hematopoiesis, hematopoietic-related transcription factors and their cofactors interact to form a complex regulatory network that controls the process of primitive hematopoiesis. Among the bHLH transcription factor family, SCL and LYL1 are key factors in embryonic hematopoiesis. SCL is responsible for initiating primitive hematopoiesis, while LYL1, a paralog of SCL, compensates for the hematopoietic impact of SCL deficiency in adulthood. However, the role of LYL1 in primitive hematopoiesis remains unclear. This study, through analysis of zebrafish blood cell scRNA-seq data, discovered high expression of CABZ01066694.1 in hematopoietic stem/progenitor cells. Sequence alignment revealed it as a short transcript of the lyl1 gene. Subsequently, using 5'RACE and sequencing, the study confirmed the existence of both long (lyl1f) and short (lyl1s) transcripts of lyl1 in zebrafish and humans, similar to mice. Further analysis of scRNA-seq and RNA-seq data from public databases showed that in zebrafish primitive hematopoietic cells, lyl1 primarily transcribes lyl1s. Finally, using Morpholino technology to knock down lyl1f and lyl1s separately, it was found that knocking down lyl1s hindered the production of primitive myeloid progenitors and primitive granulocytes, whereas knocking down lyl1f promoted the production of primitive macrophages. In conclusion, this study demonstrates the existence of long and short transcripts of lyl1 in zebrafish and humans, with distinct roles in regulating primitive myelopoiesis, providing new insights into the regulation of primitive hematopoiesis.