On the basis of fish collections made between 1991 and 2014, four species of the seasonal killifish genus Neofundulus are reported to occur in the Brazilian Pantanal, Paraguay river basin: N. parvipinnis, endemic to the Cuiabá and São Lourenço river drainages, in the northern portion of the Pantanal; N. rubrofasciatus, new species, from the Miranda river drainage, N. aureomaculatus, new species, from the Aquidauana river drainage, both in the south-eastern portion of the Pantanal; and N. paraguayensis, occurring in the Paraguay and Nabileque river floodplains, in the southern part of the Pantanal. The new species are diagnosed by unique colour patterns, and a combination of morphological characters states indicating that they are more closely related to N. parvipinnis and N. splendidus than to N. paraguayensis.

Type locality: Brazil: Estado de Mato
Grosso do Sul: Município de Aquidauana: temporary pool about 15 km NW of the town of Aquidauana, road MS-170, Aquidauana river floodplains, Paraguay river basin, 20°19’14”S 55°50’36”W, altitude about 135 m;

Max. size 7.5 cm.
Dorsal 15.3
Anal 16.3,
D/A 2.7,
LL scale count (average)37.0
Pre- dorsal length to % SL – 68.3 %
Depth to % SL – 22.0 %