Differences between sexes in growth, ecology and behaviour strongly shape species biology. In some animal groups, such as spiders, it is difficult or impossible to identify the sex of juveniles based on external morphology. This information would be useful for field surveys, behavioural experiments and ecological studies, such as those on sex ratios and dispersal. In species with sex chromosomes, sex can be determined based on the specific sex chromosome complement. Additionally, information on the sequence of sex chromosomes provides the basis for studying sex chromosome evolution. We combined cytogenetic and genomic data to identify the sex chromosomes in the sexually dimorphic spider Argiope bruennichi and designed quantitative real-time polymerase chain reaction sex markers. We found that the genome size and GC content of this spider fall into the ranges reported for the majority of araneids. The male karyotype is formed by 24 acrocentric chromosomes with an X1X20 sex chromosome system, with little similarity between X chromosomes, suggesting an origin of these chromosomes by X chromosome fission or early duplication of an X chromosome and subsequent independent differentiation of the copies. Our data suggest X chromosomes of similar sizes in A. bruennichi. They are smaller chromosomes of the complement. Our findings open the door to new directions in spider evolutionary and ecological research.