The genetic diversity of freshwater fishes is still anonymous in several drainage systems in northeast India. Moreover, the comparative genetic analysis is largely sporadic to judge their actual diversity and true status. We generated 89 DNA barcodes of 40 morphologically identified fishes collected from two major tributaries of Brahmaputra River. The comparative study revealed that most of the species were clearly discriminated by their estimated genetic distances and monophyletic clustering in Bayesian (BA) tree. Considering the genetic divergence (2%) for species discrimination boundary, the high genetic diversity (2.36–10.73%) was detected in 11 species (
Macrognathus pancalus
,
Channa punctata
,
Puntius terio
,
Bangana ariza
,
Garra arupi
,
Badis badis
,
Mystus vittatus
,
Rita rita
,
Gagata cenia
,
Mastacembelus armatus
, and
Danio dangila
), which signified the occurrence of concealed genetic diversity in this ecozone. However, the insignificant genetic distances were also noticed in few reportedly valid species:
Channa stiktos
and
C. ornatipinnis
(1.43%);
Mystus ngasep
,
M. rufescens
, and
M. carcio
(0.4%);
Glyptothorax trilineatus
,
G. churamanii
, and
G. verrucosus
(0.4%);
Botia almorhae
,
B. histrionica
,
B. lohachata
, and
B. rostrata
(0–0.4%);
Barilius barilia
and
B. vagra
(0.4%);
Batasio merianiensis
and
B. tengana
(1.2%);
Puntius chola
and
P. fraseri
(0%),
Schistura beavani
and
S. paucireticulata
(0%); hence to validate this species, generation of more barcode data was required from their types or topotypes. The present study would help to develop conservation schemes for the native species and collegiate ecosystem, which associated with the livelihoods of millions of ethnic communities in this region.