Chinese spined loach, Cobitis sinensis complex are primary benthic fishwater fishes with inhabiting on the substratum of small pebbles or coarse sand in mountain streams. In Taiwan, C. sinensis complex has been recorded from most drainages of western side of the Central Mountain Ridge including from Ilan, Taipei, even to Pingtung County. In Taiwan, all Cobitis populations have been considered as C. sinensis which types assigned from the Szechuan province, Yangtsi River basin by Sauvage and Dabry de Thiersant (1874). Due to very limited diagnostic morphological features can be detected and studied in detail, it¡¦s more important for reconstructing the molecular phylogenetic relationship by DNA sequences. We also study in the reproductive biology by the samples in Kao-ping River, and to compare the realationship by the samples in Tan-shuei River. By morphological analysis and mtDNA D-loop sequences clustering, we can recognize the C. sinensis complex of Taiwan and southern China into three group: (1) northern and middle Taiwan (NT) (as typical C. sinensis); (2) three River basins in southern Taiwan (ST) (C. sp1); (3) other samples from the Fujian provinces (ST) (C. sp2). They can be distinguished by very distinct differentiations of the scale of Canestrini. The type of NJ tree and MP tree are the overall same topology, mean that the trees of result are the stable phylogenetic tree of the whole C. sinensis complex. By the result of reproductive biological study, GSI index rise in May and drop off in September, the high peak time is close to five months. The increasing tendency of GSI is later than the temperature rising, but earlier than the rainfall begin in July. They share the similar trend among the comparison of the mature of gonad¡Bthe distribution of egg diameter and GSI index.