Pyknons are specific human/primate-specific DNA motifs at least 16 nucleotides long that are repeated in blocks in intergenic and intronic regions of the genome and can be located in a new class of non-coding RNAs of variable length. Recent studies reported that pyknon deregulation could be involved in the carcinogenesis process, including colorectal cancer. We evaluated the expression profile of a set of 12 pyknons in a set of molecularly characterized colorectal cancer (CRC) patients. The pyknons (
PYK10
,
PYK14
,
PYK17
,
PYK26
,
PYK27
,
PYK40
,
PYK41
,
PYK42
,
PYK43
,
PYK44
,
PYK83
, and
PYK90
) expression was determined by qRT-PCR. A pilot analysis of 20 cases was performed, and consistent results were obtained for
PYK10
,
PYK17
,
PYK42
,
PYK44
, and
PYK83
. Further, the expression of the selected pyknons was evaluated in 73 CRC cases. Moreover, in 52 patients, we compared the expression profile in both tumor and normal tissues. All five pyknons analyzed showed significantly lower expression levels in the tumor compared to normal tissue. It was observed an association between expression of
PYK10
with
TP53
mutations (
p
= 0.029),
PYK17
to histologic grade (
p
= 0.035), and
PYK44
to clinical staging (
p
= 0.016). Moreover, levels of
PYK44
were significantly associated with the patient's poor overall survival (
p
= 0.04). We reported the significant downregulation of pyknons motifs in tumor tissue compared with the normal counterpart, and the association of lower
PYK44
expression with worse patient outcome. Further studies are needed to extend and validate these findings and determine the clinical-pathological impact.