Pheromones, arguably the most ubiquitous mode of animal communication, are determined by both genetic and environmental factors. Recent evidence suggests that diet may be an important determinant of pheromone variation, which may both enhance and reduce the reliability of the chemical signal. We investigated experimentally the impact of population origin and diet on chemical signals used in mate assessment by monogynous males of the golden-banded orb web spider, Argiope trifasciata. Initial mate preference experiments revealed environmentally determined fine scaling of male mate choice: shortly after their introduction into the laboratory, male spiders preferred females from either their own or a nearby population rather than females from a distant population, suggesting that male choice is driven by phenotype matching, ensuring correct species identity. However, when diet was controlled, males preferred females originating from a distant rather than the same population, allowing males to choose a mate with the most potential genetic benefits. A second set of experiments clearly demonstrated that diet affected the chemical compounds on the surface of the silk threads produced by females, and that males preferred females that had experienced a similar diet. We suggest that phenotype matching strongly influences broad-scale male mating preferences, but it remains to be seen how a combination of genetic and environmental (e.g. dietary) factors influence the relative abundance of these, and perhaps other, mate choice-relevant, silk-bound chemical cues.