Asthma and chronic obstructive pulmonary disease (COPD) are now recognized to be able to co-exist as asthma–COPD overlap (ACO). It is clinically relevant to evaluate whether patients with COPD concurrently have components of asthma in primary care. This is because: (i) ACO is a relatively common condition among asthma (over 40 years of age) or COPD irrespective of its diagnosis criteria; (ii) patients with ACO can have higher frequency of exacerbation and more rapid decline in lung function than those with asthma or COPD; and (iii) asthmatic features such as eosinophilic airway inflammation are promising indicators for prediction of inhaled corticosteroidresponsiveness in COPD. The aim of this review to evaluate diagnostic markers for ACO. We searched PubMed for articles related to ACO published until 2020. Articles associated with diagnostic biomarkers were included. We identified a total of 25 studies, some of which have revealed that a combination of biomarkers such as fractional exhaled nitric oxide and serum immunoglobulin E is useful to discern type 2 inflammation in the airways of COPD. Here, we review the current understanding of the clinical characteristics, biomarkers and molecular pathophysiology of ACO in the context of how ACO can be differentiated from COPD.