Yellow drum (Nibea albiflora) is one of the most important marine fish species in China. However, the natural outbreak of red head disease has seriously restricted the aquaculture development of yellow drum, and genetic improvement has become a potential strategy. In the research, 37 DNA pools (1066 samples) were generated with Pool-seq, which significantly reduces costs. In order to correct place factors and satisfy the requirement of multi-pool joint analysis, we propose and utilize three methods to locate disease-resistant regions with 33 DNA pools. The additive property of χ2 distribution and log-likelihood ratio test of logit regression were firstly used for joint localization of disease-resistant regions; secondly, we take advantage of the modified G′ value to further narrow candidate regions. One red head disease-resistant region of 10.9 Mb–13.11 Mb on chr22 was determined. Forty-one genes (36 non-repetitive genes) were found in the region by functional gene annotation. Twelve non-repetitive genes were involved in the immune responses, where one nitr gene and several genes (i.e., tifa, alpk1, mapk1 and transcription factor p65) exist in NF-κB signaling pathway were identified, suggesting that NF-κB signaling pathway may be important in red head disease resistance.