Kinases and phosphatases are crucial for cellular processes and animal development. Various sets of resources in Drosophila have contributed significantly to the identification of kinases, phosphatases and their regulators. However, there are still many kinases, phosphatases and associate genes with unknown functions in the Drosophila genome. In this study, we utilized a CRISPR/Cas9 strategy to generate stable mutants for these unknown kinases, phosphatases and associate factors in Drosophila. For all the 156 unknown gene loci, we totally obtained 385 mutant alleles of 105 candidates, with 18 failure due to low efficiency of selected gRNAs and other 33 failure due to few recovered F0, which indicated high probability of lethal genes. From all the 105 mutated genes, we observed 9 whose mutants were lethal and another 4 sterile, most of which with human orthologs referred in OMIM, representing their huge value for human disease research. Here, we deliver these mutants as an open resource for more interesting studies.