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Freemartinism, a primary disorder of sex development (DSD) in cattle, is associated with leu - kocyte chimerism (60,XX/60,XY). The diagnosis of DSD is easy if it is known that a heifer with abnormally developed reproductive tracts originates from a heterosexual twin birth, but it is not so obvious in the case of single born calves. In the present study twelve DSD heifers which were single born (singletons) and culled due to the abnormal development of internal genitalia were studied using cytogenetic and molecular tech - niques. Among the heifers 7 appeared to be chimeric (60,XX/60,XY and the presence of the genes residing in the Y chromosome: SRY and AMELY) and 5 had a normal female karyotype (60,XX and a lack of the Y-linked genes). In addition, milk productivity was analyzed in relation to the incidence of twinning at a local Dairy Cattle Breeding Centre, from which 8 studied singletons (6 chimeric and 2 with a normal female karyotype) originated. It was found that in the years 2005-2013 an upward trend for average milk yield (from 9700 kg in 2005 to 11 500 kg in 2013) was associated with the increase of twin births (from 1.5% in 2005 to 5.9% in 2013). Our study showed that approximately 60% of single born heifers with abnormally developed internal genitalia were freemartins (a male co-twin died during pregnancy), while DSD etiology of the other cases (60,XX and a lack of the Y-linked genes) remains unknown. It cannot be excluded that some of these heifers represent a testicular/ovotesticular DSD (60,XX and SRY-negative). In conclusion, our study suggests that the occurrence of freemartins and other DSD in single born heifers seems to be an underestimated problem in cattle breeding.