To facilitate the establishment of recombinant Chinese hamster ovary (rCHO) cell lines with dihydrofolate reductase (dhfr)-mediated gene amplification, a primary selection method based on morphology of parental CHO clones has been developed. Morphology of parental clones that were made by transfecting various plasmids encoding thrombopoietin (TPO) and its analogs and humanized antibodies into dhfr-deficient (dhfr) CHO cells was not uniform. Morphology of many parental clones exhibiting high-level expression of the introduced gene was similar to that of nontransfected dhfr CHO cells. On the other hand, most parental clones with low-level expression experienced noticeable morphological changes such as bipolar fibroblast-like morphology. In case of selection of parental clones with TPO expression level higher than 200 ng/mL, morphological selection improved selection efficiency by 3.5-fold compared with random selection. Furthermore, when subjected to methotrexate for gene amplification, parental clones that were selected based on morphology elevated the expression level as much as those that were selected randomly. Taken together, morphological selection of parental clones can facilitate the establishment of rCHO cell lines expressing recombinant proteins.