Zebrafish is a powerful model for the investigation of hematopoiesis. In order to isolate novel mutants with hematopoietic defects, large-scale mutagenesis screening of zebrafish was performed. By scoring specific hematopoietic markers, 52 mutants were identified and then classified into four types based on specific phenotypic traits. Each mutant represented a putative mutation of a gene regulating the relevant aspect of hematopoiesis, including early macrophage development, early granulopoiesis, embryonic myelopoiesis, and definitive erythropoiesis/lymphopoiesis. Our method should be applicable for other types of genetic screening in zebrafish. In addition, further study of the mutants we identified may help to unveil the molecular basis of hematopoiesis.