Asparagus ( Asparagus officinalis L.) is a dioecious species, with both male and female individuals. Male plants are more desirable to cultivate than female plants because they have higher yields, and, unlike female plants, they do not have a weed problem resulting from fallen seeds. A male-specific DNA marker is currently available to identify the sex of asparagus individuals using total DNA extracted from cladodes and roots. However, no published method is currently available for DNA extraction and PCR amplification from asparagus seeds. In this study, we tested several heat-resistant DNA polymerases for PCR and several methods for extracting DNA from asparagus seeds and successfully established a method for identifying the sex of asparagus seeds using this male-specific DNA marker. We found that PCR amplification of DNA extracted from asparagus seeds using simple methods such as single-step DNA extraction requires the use of high efficiency DNA polymerase. By contrast, many types of heat-resistant DNA polymerases can be used for PCR amplification of high-quality DNA extracted from asparagus seeds using a commercially available DNA extraction kit. Our method for sex identification of asparagus seeds could facilitate quality checking of all-male asparagus seeds and accelerate the screening of super-male asparagus.