SUMMARYDinoflagellates have a rich history of characterization of their membrane‐reinforcing sterols because of a structural diversity and chemotaxonomic utility uncommon to other classes of algae. The Kareniaceae are no exception in that they produce sterols, often containing a Δ8(14) nuclear unsaturation, which are rare in most other dinoflagellate groups. A continuing goal of our laboratory is to examine the sterols of previously uncharacterized dinoflagellates to assess their sterol‐based chemotaxonomy compared to other members of the Dinophyceae. Asterodinium gracile, a member of the Kareniaceae, has not been commercially available for study until recently. To this end, our objective was to characterize the sterols of A. gracile to determine whether they match the 4α‐methyl‐substituted, Δ8(14)‐nuclear‐unsaturated sterols, such as (24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (gymnodinosterol; C29:2) and 27‐nor‐(24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (brevesterol; C28:2), of most species within the canonical genera Karenia, Karlodinium and Takayama, or instead the 4‐desmethyl sterols, such as 27‐nor‐(24R)‐23‐methyl‐ergosta‐8(14),22‐dien‐3β‐ol (C28:2), of its chemotaxonomically atypical, yet closest phylogenetic relative, Karenia papilionacea. We have observed A. gracile to produce two 4α‐methyl‐substituted sterols with the suggested structures of 4α‐methyl‐5α‐ergosta‐8(14)‐en‐3β‐ol (C29:1) and 4α‐methyl‐5α‐ergosta‐8(14),24(28)‐dien‐3β‐ol (amphisterol, C29:2), as major sterols amongst five other minor sterols, which included the common dinoflagellate sterol cholest‐5‐en‐3β‐ol (cholesterol, C27:1), but did not include gymnodinosterol or brevesterol (as in most canonical Kareniaceae) or 27‐nor‐(24R)‐23‐methyl‐ergosta‐8(14),22‐dien‐3β‐ol (as found in K. papilionacea). Detection of amphisterol and 4α‐methyl‐5α‐ergosta‐8(14)‐en‐3β‐ol is notable because they are sterols associated with some species of Amphidinium, such as Amphidinium carterae, and, to our knowledge, have not been observed in a member of the Kareniaceae before. Discovery of these major sterols in A. gracile expands our knowledge of the range of sterols produced by the Kareniaceae and indicates a shared chemotaxonomy with some species of Amphidinium.