Mesenchymal chondrosarcoma (MC) is a rare sarcoma that typically arises in adolescents and young adults and characteristically harbours a HEY1-NCOA2 gene fusion. A recent study has shown that NKX3.1 immunohistochemistry (IHC) is highly specific and sensitive in MCs. NKX3.1 is a nuclear marker expressed in prostatic tissue and is widely used in most laboratories to determine prostatic origin of metastatic tumours. In the current study we investigated whether this stain can be used in the diagnostic workup of MC, as it may assist in triaging cases for further molecular testing, by assessing its expression in a cohort of MCs and in a wide spectrum of sarcoma types. Furthermore, we aimed to elucidate if expression of NKX3.1 by MCs is related to androgen receptor (AR) expression. We identified NKX3.1 positive nuclear staining in 9 of 12 individual patients of MC (n=20 of 25 samples when taking into account separate episodes). Four of the five negative specimens had been previously subjected to acid-based decalcification. NKX3.1 was negative in 536 samples from 16 non-MC sarcomas derived from largely tissue microarrays (TMAs). Overall, we identified 80% sensitivity and 100% specificity for NKX3.1 IHC in MCs. The sensitivity increased to 95.2% when acid-based decalcified specimens were excluded from the analysis. No correlation between NKX3.1 expression and AR IHC was identified. In summary, our findings indicate that NKX3.1 nuclear positivity is highly sensitive and specific for MC, provided that ethylenediaminetetraacetic acid (EDTA)-based rather than acid-based decalcification is used for sample processing. NKX3.1 IHC in the right clinical and histopathological setting can potentially be sufficient for the diagnosis of MC, reserving molecular confirmation only for equivocal cases.