Rosa centifolia is one of the famous oil producing species of roses, used to extract oil and other products. The present study was performed at Institute of Horticultural Sciences, University of Agriculture Faisalabad (Pakistan) to evaluate the morphological and genetic diversity among different genotypes of Rosa centifolia taken from Pakistan and USA and to develop an in vitro propagation protocol for mass production of healthy plants round the year. For study morphological diversity 24 plant, leaf and flower parameters were selected from (UPOV) plant descriptor. Data of these parameters from 8 genotypes of Rosa centifolia found in 8 districts of Punjab, Pakistan and 8 genotypes found in USA was collected according to the descriptor. Young unopened leaves of these genotypes were collected for DNA extraction through CTAB method, which was used for genetic analysis by SSR markers. For in vitro propagation of Rosa centifolia young axillary buds were used as explant. Different concentrations of BAP (0.2, 0.4, 0.6 and 0.8 μM) and Kinetin (0.2, 0.4, 0.6 and 0.8 μM) alone and in combination were used for shoot formation. For root induction various concentrations of IBA (0.2, 0.4 and 0.6 μM) and NAA (0.2, 0.4 and 0.6 μM) were used. Statistical analysis was performed on the data by using SAS statistical analysis (Version 9.3) software. Principle component analysis was used to indicate the morphological diversity among 8 genotypes of Rosa centifolia in Pakistan and 8 from USA. It showed the highest phenotypic diversity between Sargodha and Sheikhupura genotypes while maximum similarity was observed between Pattoki and Faisalabad genotypes. Among 8 genotypes of Rosa centifolia from USA, maximum phenotypic association was noticed within Fantin- Latour and Rosa de Meaux while maximum diversity was between Cabbage rose 2 and Paul Ricault. During studying genetic diversity by using SSR markers genetic similarity was observed among the genotypes of one district but genotype of different districts expressed genetic diversity, which was maximum between Kahror Pakka and Pattoki genotypes but minimum between Faisalabad and Sheikhupura genotypes from Pakistan. Rosa centifolia of Pakistan and Cabbage rose 2 of USA were in the same group, which reflect genetic relationship between them. During comparison of Rosa centifolia with Rosa damascena, Pakistani genotypes of Rosa centifolia were genetically more closer to Rosa damascena genotype from Isfahan (Iran) and Pattoki (Pakistan). Rosa damascena Faisalabad was in separate group than other Rosa damascena genotypes and showed closer genetic association with Rose de Meaux and Gros Choux d’Hollande. During in vitro propagation of Rosa centifolia BAP at 0.4 μM along with 0.2 μM of Kinetin produced shoots with minimum number of days (10.20). BAP at concentration of 0.2 μM proved as prominent by producing maximum number of shoots (1.93) while 0.2 μM BAP in combination with 0.2 μM Kinetin produced shoots with maximum shoot length (5.55 cm). MS media supplemented with higher concentration (0.4 μM and 0.6 μM) of BAP along with 0.2 μM Kinetin first produced callus on the explants and after few days it also produced the shoots. In case of root induction 0.4 μM IBA proved excellent and produced roots with minimum number of days (10.07), maximum number of roots (2.47) and maximum root length (4.21 cm).