Pretreatment is one of the crucial steps in the process of plant tissue preparation to study morphology and the number of chromosomes to form the species karyotype. The research to determine the effect of pretreatment on H.rosa-sinensis chromosome L. performed from August 2016 to May 2017. Two pretreatment factors used are pretreatment agents (cold water, paradichlorobenzene (PDB), 8-hydroxyquinoline (OQ), and PDB combined with OQ (1:1)) and immersing time (3 hours, 6 hours, 12 hours and 24 hours). The shoot tip of H. rosa-sinensis crested peach used in this study. The morphology and the number of chromosomes were observed to determine the effect of pretreatment. The results showed that among the pretreatment agents used in this study, paradichlorobenzene gave the best staining results for chromosomes. Meanwhile, no significant effect of the duration of immersion to chromosomes. The size of the H.rosa-sinensis chromosome is small and does not spread. The number of chromosomes is 2n = ca 28-67 and mixoploid.