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MicroRNAs (miRNAs) are small non-coding RNAs about 20–24 nucleotides long, playing regulatory role. The conserved nature among the various organisms makes them a good source of new miRNAs discovery by comparative genomics approach using bioinformatic tools. A systematic search approach was used for inter-species homologs of miRNA precursors (pre-miRNAs), from known
Helianthus
expressed sequence tags (ESTs). The study resulted in 61 novel miRNAs belonging to 34 families from
Helianthus
ESTs. The 28 miRNA families; mir 159,160, 164, 170, 390, 393, 413, 415, 419, 426, 446, 530, 822, 842, 846, 1310, 1888, 2086, 2657, 2667, 2678, 2659, 2911, 2938, 3440, 3521, 3623, and 3630 are reporting for the first time in
Helianthus
. In the 61 new miRNAs, 20 are from
H.
tuberosus
, 17 miRNAs belong to
H.
annus
, 8 are from
H.
ciliaris
, 5 to
H.
exilis
, 4 is from
H.
argophyllous
,
H.
petiolaris
each and 3 are from
H.
paradoxus
. All the pre-miRNAs form stable minimum free energy (mfe) stem-loop structure as their orthologues form and the mature miRNAs reside in the stem portion of the stem-loop structures. Their targets consist of growth and development related, transcription factors, signalling pathway kinases, stress resistant proteins and transport related proteins.