Non-obstructive azoospermia (NOA) is a complex and severe condition whose etiology remains largely unknown. In a genome-wide association study (GWAS) of NOA in Chinese men, few loci reached genome-wide significance, although this might be a result of genetic heterogeneity. Single nucleotide polymorphisms (SNPs) without genome-wide significance may also indicate genes that are essential for fertility, and multiple stage validation can lead to false-negative results. To perform large-scale functional screening of the genes surrounding these SNPs, we used in vivo RNA interference (RNAi) in Drosophila, which has a short maturation cycle and is suitable for high-throughput analysis. The analysis found that 7 (31.8%) of the 22 analyzed orthologous Drosophila genes were essential for male fertility. These genes corresponded to nine loci. Of these genes, leukocyte-antigen-related-like (Lar) is primarily required in germ cells to sustain spermatogenesis, whereas CG12404, doublesex-Mab-related 11E (dmrt11E), CG6769, estrogen-related receptor (ERR) and sulfateless (sfl) function in somatic cells. Interestingly, ERR and sfl are also required for testis morphogenesis. Our study thus demonstrates that SNPs without genome-wide significance in GWAS may also provide clues to disease-related genes and therefore warrant functional analysis.