Increasingly, conservation genetics pinpoint the use of biological matrices collected without stressing wildlife. Cervid’s antlers seem to fit with this need. We verified the amplification success rate from DNA obtained from red deer antlers collected in the State Nature Reserve of Bosco della Mesola, Northern Italy and its use for conservation purposes. Here occurs the only native red deer population of peninsular Italy, recently recognized as a distinct subspecies (Cervus elaphus italicus). Four antlers stored at room temperature for four years and four samples highly degraded by environmental conditions were analyzed using a multimarker approach. We utilized a simple, inexpensive method to extract DNA from drilled antlers powder. This study confirms that weathered antlers can be a suitable source of DNA also in Mediterranean climate characterized by strong seasonal fluctuations, and not only in dry climates. Our results pointed out that only burr drilling yielded good quality amplifiable DNA. Antlers can be used in particular for molecular genetic studies on rare or threatened species of cervids as providing an efficient and cost-effective non-invasive sampling.