Citrus black spot is an important fungal disease of citrus resulting in fruit drop and rind blemish in tropical and subtropical production areas. The disease is incited by the fungus Phyllosticta citricarpa (McAlpine) van der Aa (synonym: Guignardia citricarpa Kiely), with control currently relying on the application of fungicides. Because the presence and expression of resistance is poorly understood, we sought to develop a method for inoculating fruit in the field that gives reproducible symptoms of citrus black spot consistent with natural field infection. We subsequently validated this method by screening 49 citrus accessions and characterized their qualitative expression of citrus black spot symptoms. Challenge inoculations were undertaken with a known isolate of P. citricarpa, and control fruit were inoculated with water or the endophyte P. paracapitalensis Guarnaccia & Crous. Our results showed that all mandarin, sweet orange, lemon and papeda types were susceptible; pummelo, lime, and sour orange types expressed immunity; while various hybrids were susceptible, resistant and immune. Hybrid progeny from crosses using pummelo [Citrus maxima (Burm.) Merr.] as a parent showed preliminary evidence of segregation for citrus black spot immunity. The implications of these results to achieve genetic improvement for citrus black spot resistance in citrus breeding programs are discussed.