A novel online system including two-dimensional liquid chromatography coupled to high-resolution mass spectrometry (2D-LC/MS) was developed and applied for comprehensive phospholipid (PL) and sphingomyelin (SM) profiling of dorsal hippocampus (DHPC), ventral (VHPC) and prefrontal cortex (PFC) brain regions in a mouse model of anxiety disorder. In the first dimension, lipid classes were distinguished by hydrophilic interaction liquid chromatography (HILIC), while the second dimensional separation of individual PL and SM species was achieved by reversed-phase (RP) chromatography. For the enrichment of lipid species in diluted HILIC effluent, two RP trapping columns were used separately. The developed fully-automated 2D method allowed the quantitative analysis of over 150 endogenous PL and SM species in mouse brain regions within 40 min. The developed method was applied in a pilot study, which aimed to find alteration of PL and SM composition in a mouse model of anxiety disorder. In the case of 37 PL and SM species, significant differences were observed between high anxiety-related behavior (AX) and low anxiety-related behavior (nAX) mice. In mice having elevated anxiety, the most typical trend was the downregulation of PL species, in particular, in VHPC.