Aims: The investigation aimed to examine the crude oil-contaminated soil Streptomyces flora and study their capability to grow on diesel fuel as a sole carbon source and their analysis for the presence of alkane hydroxylase gene (alkB) by PCR. Methodology and results: A total of 17 Streptomyces isolates were recovered from hydrocarbon-contaminated soil samples on starch casein nitrate agar medium with the ability of 4 isolates to grow on diesel [0.1 % (v/v)] as assessed by agar plate diffusion method, enzymatic assay and dry weight measurements. The ability of the four isolates (JR2b, JR3a, JR5b, and JR6f) to grow on diesel was revealed by the colour change of the reaction mixture and showing a growth response by growing around diesel-containing wells with a percentage increase in the dry weight of 24.60, 26.23, 18.03, and 18.03 after 28 days of incubation as compared to zero time, respectively. Although the four isolates were capable to degrade diesel as indicated by the three assessment techniques, they did not show any PCR product. Conclusion, significance and impact of study The isolates that grew on diesel and showed no PCR product might not contain the alkB gene, which implies that alkB gene is not the only gene that is responsible for the degradation of alkanes.