Generally, commercial P-TREF (preparative temperature rising elution fractionation) equipment carries out an experiment for polyolefin samples between room temperature (or the lowest temperature at 20 °C) and 150 °C. Although we have applied the traditional TREF method in polyethylene and propylene resin in our previous studies, it failed to fractionate poly(1-butene) with a polymorph structure. Up to now, it has been a challenge to realize an effective P-TREF separation procedure for poly(1-butene) resins. Based on the analysis and experiments, it was found that a low temperature was important for poly(1-butene) resin. Herein, a novel P-TREF instrument with a broad temperature range from −80 to 150 °C is designed and fabricated. By applying this equipment, one poly(1-butene) copolymer, i.e., 1-butene/ethylene random copolymer, is first effectively fractionated according to its crystallizability by the TREF principle. The main fractions are eluted at 30, 42, and 46 °C, corresponding to the weight percent of 12.81%, 18.05%, and 39.52%, respectively. Chain microstructures of the original resin and its fractions are further characterized by high-temperature gel permeation chromatography coupled with triple detectors (refractive index−laser light scattering−viscometer), 13 C-nuclear magnetic resonance spectroscopy ( 13 C NMR), and differential scanning calorimetry (DSC). With an increasing elution temperature from −30 to 46 °C, the ethylene content of the fractions decreases from 12.6 to 2.9 mol %; meanwhile, the isotacticity increases from 41.2% to 83.6%, and the crystallinity increases gradually. From these results, the TREF technique can be extended to resins (for example, poly(1-butene) copolymer) with slow crystallization kinetics, and the chain microstructure of polymorphic poly(1-butene) copolymer can be analyzed in detail. This will lay a foundation for both basic research and industry applications.