Banana (genus Musa) is an important economic and food crop. Banana plants are attacked by many bacterial, fungal and viral pathogens. One of the most important viral pathogens of banana is the Banana bunchy top virus (BBTV), which causes the banana bunchy top disease (BBTD). In Pakistan banana industry has faced the problem of BBTV since late 1980s, when a severe epidemic caused drastic economical loses. The genome of BBTV comprises at least six integral components of circular single stranded DNA (cssDNA) molecules, each about 1 Kb in size. BBTV belongs to genus Babuvirus in Nanoviridae, one of the three cssDNA virus families. It is transmitted by black banana aphid Pentalonia nigronervosa Coq. Research work on BBTV of Pakistani origin was fairly limited when this work was started. Therefore need existed to study the virus isolates infecting the banana crop in Pakistan and analyze their phylogenetic relationships with other isolates and cssDNA viruses for comprehensive genetic characterization. As no known source of resistance to BBTV is available in nature, hence to control this disease, creation of artificial viral resistance seems to be the only available strategy. This can be achieved by gene silencing technologies like RNA interference (RNAi) which is very potent and has great potential to be utilized against BBTV. This technology requires the complete knowledge of genome sequence of the target virus and genetic variability associated with its population. To characterize BBTV from Pakistan, the DNA-R of an isolate from Tandojam area (TJ1 isolate) was amplified by PCR, cloned and sequenced. The phylogenetic analysis of TJ1 isolate suggested that it belongs to the South Pacific sub-group of BBTV. Later, primers were designed for two rounds of amplification of other components. The PCR assay was optimized for amplification of all the components simultaneously along with actin gene as positive control. This first time reported multigene PCR assay is valuable tool for detection of BBTV in planting material. The amplified products were cloned and sequenced, and analyzed together with the other sequences available in the GenBank. The sequence analysis of common region stem-loop (CR-SL) and the significant variation in component population indicated that the master rep (M-Rep) of BBTV may not only initiate and terminate the replication but also regulate the replication of the genomic components. The analysis of common region major (CR-M) revealed its clustering according to the sub-groups, which correlates well with its proposed function i.e. conversion of cssDNA into transcriptionally active double stranded DNA (dsDNA). The analysis of the genomic components revealed two sub-groups of BBTV. On the basis of all six components, it was found that Pakistani isolates belong to the South Pacific sub-group. The sequence analysis of genomic components showed different level of sequence conservation indicating that they are under different extent of evolutionary pressure. The multigene phylogenetic analysis of BBTV and other cssDNA viruses showed that the members of Nanoviridae share common ancestral relationships among themselves as well as with Geminiviridae and Circoviridae. The study indicated a low level of genetic variability associated with the BBTV population in Pakistan, estimated by sequencing DNA-R and DNA-S of thirteen isolates originating from different parts of the Sindh province. The DNA-R is more conserved component compared to DNA-S. The low level of genetic variability has great potential for use of molecular approaches to create the artificial resistance against this virus. Therefore DNA-R; the most conserved and vital component for BBTV life cycle being involved in the replication of other components, was used to develop an RNAi based vector against the M- Rep coding region. A GFP based reporter construct was also developed which can be used in monitoring of the RNAi based silencing of M-Rep in easily transformable non-host plants. This novel strategy for confirmation of silencing alleviates the need to work only in the ultimate host. This strategy may also be applicable to other viruses. The RNAi and reporter vectors are available for use in banana transformation programs to create genetic resistance in banana against BBTV. The analysis of isolates belonging to the South Pacific sub-group indicated that the Pakistani isolates have close relationships with the Indian, Egyptian and Australian isolates. However, due to differences in component specific phylogenies and non-availability of sequences of all the components of various reported virus isolates from the South Pacific sub- group, it is not prudent to construct a comprehensive picture of the origin of disease in Pakistan on molecular basis. Conclusively, during this study, complete sequence of DAN-R of TJ1 isolate from Pakistan and its sub-group was published for the first time*. The study also includes sequencing of the complete genome of TJ1 isolate, and establishes the phylogenetic relationships with other BBTV isolates. Based on multigene and additional Rep analysis, the phylogenetic relationships of cssDNA virus families were determined which indicates the possibilities of genetic material exchange between these families. This study further reports the genetic variability of BBTV population in Pakistan based on the full-length sequences of DNA-R and DNA-S of thirteen isolates from different locations of the Sindh province. The information generated during this work were utilized to develop an RNAi construct against the most conserved and important protein, the M-Rep along with its reporter vector. xvii Nevertheless a comprehensive picture of genetic variability may need to be extended to remaining components of the reported and other isolates. The construct designed in this study needs to be transformed into banana plants to explore its effectiveness. The RNAi constructs based on the similar strategy may be developed for other components/proteins of BBTV to exploit the possibility of multigene silencing against this virus. *Hyder, M. Z., S. Q. Raza, S. Hameed, S. Kahlid and S. M. S. Naqvi. 2007. Phylogenetic relationship of TJ1 isolate of Banana bunchy top virus from Pakistan by DNA-R sequence analysis. Can. J. Plant. Pathol., 29: 63-68.