This study presents development of a test method for characterization of interlayer, mode-I fracture toughness of fused filament fabrication (FFF) materials using a modified double cantilever beam (DCB) test. This test consists of DCB specimen fabricated from using unidirectional FFF layers, an 8 μm Kapton starter crack inserted in the midplane during the printing process, and reinforcing glass/epoxy doublers to prevent DCB arm failure during loading. DCB specimens are manufactured with a commercially available 3D printer using unreinforced Acrylonitrile Butadiene Styrene (ABS) and chopped carbon-fiber-reinforced ABS (CF-ABS) filaments. To examine the effect of the FFF printing process on fracture toughness, additional ABS and CF-ABS specimens are hot-press molded using the filament material, and tested with the single end notch bend (SENB) specimen configuration. The fracture toughness data from DCB and SENB tests reveal that the FFF process significantly lowers the mode-I fracture toughness of ABS and CF-ABS. For both materials, in situ thermal imaging and post-mortem fractography shows, respectively, rapid cool-down of the rasters during filament deposition and presence of voids between adjacent raster roads; both of which serve to reduce fracture toughness. For CF-ABS specimens, fracture toughness is further reduced by inclusion of poorly wetted chopped carbon fibers. Although this study did not attempt to optimize the fracture performance of FFF specimens, the results demonstrate that the proposed methodology is suitable for design and optimization of FFF processes for improved interlayer fracture performance.