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Abstract: MicroRNAs (miRNAs) are small non-coding RNAs, which are known to regulate plant responses to abiotic stresses, such as Cold Stress (CS) and during normal growth and development. In Brassica napus (canola), miRNAs regulate various developmental processes and responses to metal stress however; their role in response to CS is largely unknown. In this study, we investigated CS induced changes in electrolyte leakage, malondialdheyde (MDA), antioxidant enzymes and photosynthetic efficiency in spring canola seedlings exposed to 4°C. Using small RNA sequencing, 70 known and 126 novel miRNAs were identified in CS leaf tissues and, among these, 25 known and 104 novel miRNAs were observed to be differentially expressed. Quantitative real-time (qRT- PCR) analysis of eight selected miRNAs confirmed their CS responsiveness. Furthermore, the expression of six out of eight miRNAs exhibited an opposite trend in a winter variety of canola, ‘Mendel’, when compared to ‘DH12075’ which might be a reflection of their cold susceptibility /tolerance. One of the miRNAs which was observed to be differentially expressed in in B.napus in response to CS was miR395. In order to further investigate its role in CS, we cloned the precursor of miR395f from B. napus, constitutively overexpressed it in Arabidopsis thaliana. Compared with the WT, A. thaliana plants overexpressing the precursor of bna pre-miR395f displayed a hypersensitive phenotype to freezing stress (-5°C) and CS (4 °C). Increased electrolyte leakage, enhanced MDA content and higher level of staining for reactive oxygen species (ROS) was observed in transgenic lines indicating altered sensitivity to cold in these pre-miR395f overexpressing lines. Analysis of expression of 18 genes related to sulfur metabolism and those for antioxidant enzymes revealed 14 transcripts to be increased after CS in transgenic lines indicating that both sulfur transport and metabolism as well as the status of sulfur containing antioxidant systems may be altered in the transgenic plants. In addition, we carried out a study to investigate genetic diversity in 64 accessions of Brassica species, including spring B. napus, winter B. napus, winter B. rapa and Recombinant Inbred Lines (RILs) generated from winter × spring B. napus crosses using B. napus miRNA-SSR markers. In total, 25 miR-SSR markers were mined from 90 known B. napus miRNA coding genes. These markers were able to distinguish the Brassica lines into five different clusters based on their taxonomic classification and growth habit. All 25 miR-SSRs were found to be polymorphic in the population,however, only the marker miR159-SSR was able to differentiate the winter and the spring growth habit types. These miR-SSR markers exhibited high polymorphism, and grouping of the Brassica accessions by cluster analysis was generally consistent with known pedigree suggesting the usefulness of this type of markers for use in breeding and research.