Red, orange and yellow carotenoid-based ornaments, which are widely used as sexual signals in many birds, fish and reptiles, are known to exhibit multidimensional chromatic variation as a result of both the concentration and relative proportions of different constituent carotenoids with differing spectral properties. This is thought to reflect intrinsic variation in signaller quality, making it a useful basis for female choice. However, whether females are able to discriminate relevant variation in carotenoid concentration and/or composition independently of each other, and of other phenotypic or behaviour traits, and if so, how this mediates their choice, is poorly understood. Here, female three-spined sticklebacks (Gasterosteus aculeatus) were presented with computer-animated courting males that varied exclusively in the appearance of their carotenoid-based colouration; specifically, each male’s signal provided a perceptual match for carotenoid colouration expressed by live males with known underlying carotenoid content, thereby providing a biologically-relevant signal while precluding confounding traits influencing female choice. Females were able to discriminate between prospective mates solely on the basis of perceived variation in the allocation of carotenoids to males’ sexual signals, and exhibited a strong preference for males with colouration indicative of higher concentrations of carotenoids in their signal, rather than in response to perceived variation in the relative proportion of constituent carotenoids. This has important implications for our understanding male signalling strategies and the information content of carotenoid-based sexual signals.