MicroRNA (miRNA) expression is deregulated in many tumors including chronic lymphocytic leukemia (CLL). Although the particular mechanism responsible for their aberrant expression is not well characterized, the presence of mutations and single nucleotide polymorphisms (SNP) in miRNA genes, possibly affecting their secondary structure and expression, has been described. In CLL, however, the impact and frequency of such variations have yet to be elucidated. Using a custom resequencing microarray, we screened sequence variations in 109 cancer related pre-miRNAs in 98 CLL patients. Additionally, the primary regions of miR-29b-2/29c were analyzed by Sanger sequencing in another cohort of 213 CLL patients. Altogether, we describe 6 novel miR-sequence variations and the presence of SNPs (n=27), most of which changed the miR-secondary structure. Moreover, some of the identified SNPs have a significantly different frequency in CLL when compared to a control population. Additionally, we identified a novel variation in miR-16-1 that had not been previously described in CLL patients. We show that this variation affects the expression of mature miR-16-1. We also show that the expression of another miRNA with pathogenetic relevance for CLL, namely miR-29b-2, is influenced by the presence of a polymorphic insertion which is more frequent in CLL than in a control population. Altogether, these data suggest that sequence variations may occur during CLL development and/or progression.