No information regarding pathogenic variability among different isolates of Ralstonia solanacearum infecting chili from different agro-ecological zones of Pakistan with varying climatic and edaphic factors is available. Therefore, in the present study, variations were observed in biovar distribution, hypersensitive response, growth and virulence among 114 isolates of R. solanacearum collected from eight agro-ecological zones of Pakistan. Out of 114 R. solanacearum isolates, 81% were identified as Biovar III, while the remaining 19% were recognized as Biovar IV. Of all the 114 isolates of R. solanacearum, 77% showed positive hypersensitive response and mucoid growth, while 23% isolates gave negative hypersensitive response with non-mucoid growth. Out of 114 isolates of R. solanacearum consisting of Biovar III and IV, 22.8% were found avirulent, 25.4% weakly virulent, 29.8% virulent, and the remaining 21.9% were highly virulent. Variations among 114 R. solanacearum isolates were also observed in four provinces of the country. Among 92 R. solanacearum Biovar III isolates, 21.7% were identified as avirulent, 25% weakly virulent, 34.4% virulent, and 22.8% were highly virulent in the eight agro-ecological zones of the country. Similarly, out of 22 R. solanacearum Biovar IV isolates, 27.3% were detected as avirulent, weakly virulent and virulent, while 18.2% isolates were found highly virulent. The isolates having non-mucoid growth and negative hypersensitive response were found avriulent, while those with mucoid growth and positive hypersensitive response were weakly virulent to highly virulent. The information will help design control strategies accordingly and develop resistant cultivars against the bacterium.