Purpose: Layer-specific global longitudinal strain (GLS) may provide important insights in patients with suspected coronary artery disease (CAD). We aimed to investigate the association between layer-specific GLS and coronary artery calcium score (CACS) in patients suspected of CAD. Methods: We performed a retrospective study of patients suspected of CAD who underwent both an echocardiogram and cardiac computed tomography (median 42 days between). Layer-specific (endocardial-, whole-layer-, and epicardial-) GLS was measured using speckle tracking echocardiography. We assessed the continuous association between layer-specific GLS and CACS by negative binomial regression, and the association with high CACS (≥400) using logistic regression. Results: Of the 496 patients included (mean age 59 years, 56% male), 64 (13%) had a high CACS. Those with high CACS had reduced GLS in all layers compared to those with CACS < 400 (endocardial GLS: −20.5 vs. −22.7%, whole-layer GLS: −17.7 vs. −19.4%, epicardial GLS: −15.3 vs. −16.9%, p <.001 for all). Negative binomial regression revealed a significant continuous association showing increasing CACS with worsening GLS in all layers, which remained significant after multivariable adjustment including SCORE chart risk factors. All layers of GLS were associated with high CACS in univariable analyses, which was consistent after multivariable adjustment (endocardial GLS: OR = 1.11 (1.03–1.20); whole-layer GLS: OR = 1.14 (1.04–1.24); epicardial GLS: OR = 1.16 (1.05–1.29), per 1% absolute decrease). Conclusion: In this study population with patients suspected of CAD and normal systolic function, impaired layer-specific GLS was continuously associated with increasing CACS, and decreasing GLS in all layers were associated with presence of high CACS.