Abstract: All-male yellow catfish (Pelteobagrus fulvidraco) and hybrid yellow catfish (P. fulvidraco♀×P. vachelli♂) are the main breeding varieties of yellow catfish. The “white spot disease” caused by Ichthyophthirius multifiliis (Ich) is one of the main diseases of yellow catfish in China. Ich resistance assay was performed on all-male and hybrid yellow catfish, as well as the darkarbel catfish (P. vachelli). By bioinformatics analysis, we identified an antimicrobial peptide HBβ-C from the skin mucus proteome of yellow catfish, a 33 amino acid peptide derived from the C terminal of hemoglobin β chain (HBβ). The anti-parasitic test indicated that HBβ-C effectively killed the trophont, tomont and theront, especially in trophont stage, and 15 µg/mL HBβ-C killed all trophont within 3min. Gene expression analysis showed that the expression of HBβ mRNA in gill and skin of hybrid yellow catfish was higher than that of all-male yellow catfish. Ich challenge significantly increased HBβ mRNA level in all-male yellow catfish, which was higher than that of hybrid yellow catfish. Interestingly, the HBβ protein level in gill of all-male yellow catfish was higher than that in hybrid yellow catfish. Immunofluorescence results showed that the antimicrobial peptide HBβ-C was specifically expressed in red blood cells. After Ich infection, HBβ-C was secreted and attached to the trophont in skin and gill. Thus, our results revealed that all-male yellow catfish was much more resistant to Ich than hybrid yellow catfish. The high HBβ protein in all-male yellow catfish and its ability to kill all stages of Ich might be closely associated with the resistance of all-male yellow catfish to Ich.