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[Display omitted] • OLE-LC–Qtof-MS is an alternative tool for spatial-resolved metabolomics. • The ranking style of fragment ion intensity was used for isomeric identification. • 46 and 41 ingredients were observed and identified from FV and JN petals. • 87 compounds exhibited regio-specific occurrence and accumulation. MS imaging (MSI) is a powerful technique for investigating the spatial distribution of metabolites in complex biological samples. However, due to the absence of liquid chromatography (LC) separation in routine MSI analysis, matrix effect is obvious and isomers identification remains challenging. To overcome these shortcomings of classical MSI tools (e.g. , DESI-MSI and MALDI-MSI) for isomer differentiation and insufficient datapoints for quantification, online extraction-liquid chromatogram–hybrid triple quadrupole-time-of-flight mass spectrometry (OLE-LC–Qtof-MS) platform has been developed for spatial metabolome. As a proof-of-concept, two species flowers namely Forsythia viridissima (FV) and Jasminum nudiflorum (JN) that bloom in early spring were collected, dried, and cut into small pieces (1.0 mm × 1.0 mm). All pieces successively underwent OLE-LC–Qtof-MS measurements. As a result, 46 and 41 metabolites were observed and identified from FV and JN petals, respectively. Particularly, each compound corresponded to a chromatographic peak and isomeric differentiation was achieved amongst a set of chlorogenic acid derivatives. The peak areas of high intensity metabolites were aligned and combined within either species. The datasets were individually converted into heatmaps for all compounds, 87 ones in total, and each grid of any heatmap was assigned to the original location in the petal. Then, the spatial-resolved distribution style of each compound crossing the petal was reflected by the re-organized heatmap bearing the petal shape. As expected, regio-specific occurrence and accumulation were observed for several compounds, particularly among the chlorogenic acid isomers. Above all, OLE-LC–Qtof-MS is an alternative tool for spatial-resolved metabolome attributing to the advantages of isomeric separation and reliable quantification. [ABSTRACT FROM AUTHOR]