Background: Coagulase-negative staphylococci (CoNS) are one of the most common contaminant microorganisms isolated from blood cultures. Few studies exploring the use of Gram staining to distinguish between Staphylococcus aureus (SA) and CoNS have been reported. Here, this study aimed to explore whether morphological features of Gram staining could identify SA or CoNS.Methods: This study was conducted at St. Luke’s International Hospital from November 2016 to September 2017. The positive blood cultures for which the Gram staining showed gram-positive cocci (GPC) in clusters were included in our study. The direct smear of Gram staining obtained from positive blood culture bottles were examined within 24 hours of positivity. We have identified and characterized the following two signs: “four-leaf clover (FLC)” if 4 GPC gathered like a planar four-leaf clover and “grapes” if the GPC gathered like grapes in a three-dimensional form. The number of fields with FLC and grapes signs in 10 fields per slide with ×1,000 power was counted, and the results in a total of 20 fields with ×1,000 power were combined. We performed a logistic regression analysis to assess whether these signs could serve as factors distinguishing between SA and CoNS. The predictive ability of these signs was evaluated based on the sensitivity, specificity, positive predictive value, and negative predictive value for CoNS via receiver operating curve analysis.Results: In total, 106 blood cultures for which Gram staining showed GPC in clusters were examined; 46 (43%) were SA, and 60 (57%) were CoNS samples. The result of multivariate logistic regression analysis showed that the FLC sign was a statistically significant marker of CoNS with an odds ratio of 1.31 (95 % confidential interval (CI): 1.07–1.61, p<0.05). In aerobic bottles, sensitivity, specificity, positive predictive value, and negative predictive value for CoNS were 0.67, 0.91, 0.92, and 0.65, respectively, and the value of area under the curve was 0.79 (95% CI: 0.67–0.91).Conclusions: To our knowledge, this is the first study to show that the FLC could be a rapid and useful indicator to identify CoNS in aerobic bottles. Thus, the presence of FLC sings could help clinicians to suspect the possibility of CoNS before the final identification by cultures.